Synthesis of clasto-lactacystin beta-lactone and analogs thereof

ABSTRACT

The present invention is directed to an improved synthesis of clasto-lactacystin-β-lactone, and analogs thereof, that proceeds in fewer steps and in much greater overall yield than syntheses described in the prior art. The synthetic pathway relies upon a novel stereospecific synthesis of an oxazoline intermediate and a unique stereoselective addition of a formyl amide to the oxazoline. Also described are novel clasto-lactacystin-β-lactones, and analogs thereof and their use as proteosome inhibitors.

BACKGROUND OF THE INVENTION

[0001] This application claims priority to U.S. provisional patentapplication Serial No. 60/055,848, filed on Aug. 15, 1997, and U.S.provisional patent application Serial No. 60/067,352, filed on Dec. 3,1997, which applications are hereby incorporated by reference.

[0002] 1. Field of the Invention

[0003] The invention relates generally to methods for preparinglactacystin and related compounds, to novel analogs of lactacystin andclasto-lactacystin β-lactone, and their uses as proteasome inhibitors.

[0004] 2. Description of Related Art

[0005] The Streptomyces metabolite lactacystin (1) inhibits cell cycleprogression and induces neurite outgrowth in cultured neuroblastomacells (Omura et al., J. Antibiotics 44:117 (1991); Omura et al., J.Antibiotics 44:113 (1991); Fenteany et al., Proc. Natl. Acad. Sci. (USA)91:3358 (1994)). The cellular target mediating these effects is the 20Sproteasome, the proteolytic core of the 26S proteasome, which is thecentral component of the ubiquitin-proteasome pathway for intracellularprotein degradation. Mechanistic studies have established thatlactacystin inhibits the proteasome through the intermediacy of theactive species, clasto-lactacystin β-lactone (2), which specificallyacylates the N-terminal threonine residue of the proteasome X/MB1subunit (Fenteany, et al., Science 268:726 (1995); Dick et al., J. Biol.Chem. 271:7273 (1996)). Lactacystin analogs are disclosed by Fenteany etal. (WO 96/32105).

[0006] The ubiquitin-proteasome pathway is involved in a variety ofimportant physiological processes (Goldberg et al., Chemistry & Biology2:503 (1995); Ciechanover Cell 79:13 (1994); Deshaies, Trends CellBiol.5:43 1 (1995)). In fact, the bulk of cellular proteins arehydrolyzed by this pathway. Protein substrates are first marked fordegradation by covalent conjugation to multiple molecules of a smallprotein, ubiquitin. The resultant polyubiquitinated protein is thenrecognized and degraded by the 26S proteasome. Long recognized for itsrole in degradation of damaged or mutated intracellular proteins, thispathway is now also known to be responsible for selective degradation ofvarious regulatory proteins. For example, orderly cell cycle progressionrequires the programmed ubiquitination and degradation of cyclins. Theubiquitin-proteasome pathway also mediates degradation of a number ofother cell cycle regulatory proteins and tumor suppressor proteins(e.g., p21, p27, p53). Activation of the transcription factor NF-κB,which plays a central role in the regulation of genes involved in theimmune and inflammatory responses, is dependent upon ubiquitination anddegradation of an inhibitory protein, IκB-α (Palombella et al., WO95/25533). In addition, the continual turnover of cellular proteins bythe ubiquitin-proteasome pathway is essential to the processing ofantigenic peptides for presentation on MHC class I molecules (Goldbergand Rock, WO 94/17816).

[0007] The interesting biological activities of lactacystin andclasto-lactacystin β-lactone and the scarcity of the natural materials,as well as the challenging chemical structures of the molecules, havestimulated synthetic efforts directed toward lactacystin and relatedanalogs. Corey and Reichard J. Am. Chem. Soc. 114:10677 (1992);Tetrahedron Lett. 34:6977 (1993)) achieved the first total synthesis oflactacystin, which proceeded in 15 steps and 10% overall yield. The keyfeature of the synthesis is a stereoselective aldol reaction of acis-oxazolidine aldehyde derived from N-benzylserine to construct theC(6)-C(7) bond. In the synthesis reported by (Uno et al., J. Am. Chem.Soc. 116:2139 (1994)), stereo selective Mukaiyama-aldol reaction of abicyclic oxazolidine silyl enol ether intermediate derived fromD-pyroglutamic acid is employed in C(5)-C(9) bond construction. Thissynthesis proceeds in 19 steps and 5% overall yield. Aldol reactionsunder basic conditions of a similar bicyclic oxazolidine intermediateform the basis of model studies reported by (Dikshit et al., TetrahedronLett. 36:6131 (1995)).

[0008] Aldol reactions of oxazoline-derived enolates feature prominentlyin the synthesis of lactacystin reported by Smith and coworkers (Suazukaet al., J. Am. Chem. Soc. 115:5302 (1993); Nagamitsu et al., J. Am.Chem. Soc. 118:3584 (1996)) and in the synthesis of (6R)-lactacystinreported by (Corey and Choi Tetrahedron Lett. 34:6969 (1993)); ChoiPh.D., Thesis, Harvard University, 44 (1995). In the former synthesis,which proceeds in 20 steps and 9% overall yield, the enolate iscondensed with formaldehyde to install a single carbon atom, which mustthen be elaborated in a number of additional steps. In the Corey andChoi synthesis, the aldol reaction selectively provides the product ofundesired stereochemistry, resulting in the eventual preparation of theC(6) epimer of lactacystin, which is devoid of biological activity.

[0009] Lactacystin has also been prepared in 22 steps and 2% overallyield from D-glucose (Chida et al., J. Chem. Soc., Chem. Commun. 793(1995)). The biosynthetic pathway involved in production of the naturalproduct has been investigated in feeding experiments involving¹³C-enriched compounds (Nakagawa et al., Tetrahedron Lett. 35:5009(1994)).

[0010] The reported syntheses of lactacystin are lengthy and proceed inlow yield. Furthermore, none of these syntheses is readily adapted foranalog synthesis. Thus, there is a need for improved methods forpreparing lactacystin, clasto-lactacystin β-lactone, and analogs thereof

SUMMARY OF THE INVENTION

[0011] A first aspect of the present invention relates to a process forforming lactacystin or analogs thereof having Formula VI orclasto-lactacystin β-lactone or analogs thereof having Formula VII:

[0012] wherein

[0013] R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl,aralkyl, where the ring portion of any of said aryl, aralkyl, or alkarylcan be optionally substituted;

[0014] R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy,alkoxyalkyl, or amido, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; and

[0015] R⁷ is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl,aryl, aralkyl or alkaryl can be optionally substituted.

[0016] A second aspect of the present invention is directed to a methodof forming formyl amides of Formula XIV:

[0017] where R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy,hydroxy, alkoxyalkyl, or amido, where the ring portion of any of saidaryl, aralkyl, or alkaryl can be optionally substituted; and

[0018] R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶when taken together with the nitrogen atom to which they are attachedform a 5- to 7-membered heterocyclic ring, which may be optionallysubstituted, and which optionally may include an additional oxygen ornitrogen atom.

[0019] A third aspect of the present invention relates to formingtri-substituted oxazolines of Formula Ia or Ib:

[0020] where R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl,aralkyl, where the ring portion of any of said aryl, aralkyl, or alkarylcan be optionally substituted; and R⁴ is aryl or heteroaryl, either ofwhich may be optionally substituted. The tri-substituted oxazolines ofFormulae Ia and Ib are useful as starting materials in forminglactacysin, clasto-lactacystin β-lactone or analogs thereof via theprocess described herein.

[0021] A fourth aspect of the present invention is directed tolactacysin, clasto-lactacystin β-lactone or analogs of Formulae VI andVI that possess unexpected biological activity. Lactacystin,clasto-lactacystin β-lactone, and analogs thereof possess biologicalactivity as inhibitors of the proteasome. They can be used to treatconditions mediated directly by the function of the proteasome, such asmuscle wasting, or mediated indirectly via proteins which are processedby the proteasome, such as the transcription factor NF-κB.

[0022] A fifth aspect of the present invention relates to pharmaceuticalcompositions, comprising a compound of Formula VI or Formula VII, and apharmaceutically acceptable carrier or diluent.

[0023] A sixth aspect of the present invention relates to methods ofinhibiting proteasome function or treating a condition that is mediateddirectly or indirectly by the function of the proteasome, byadministering a compound of Formula VI or Formula VII that possessesunexpectedly high activity in inhibiting the proteasome. PreferredEmbodiments are directed to the use of a compound of Formulae VI or VIIto prevent or reduce the size of infarct after vascular occlusion forexample, for treating neuronal loss following stroke. An additionalpreferred embodiment is directed to the use of said compounds fortreating asthma.

[0024] A seventh aspect of the invention relates toenantiomerically-enriched compositions of formyl amides of Formula XIV.

[0025] An eighth aspect of the present invention relates to novelindividual intermediates, such as aldols of Formula II and aminodiols ofFormula III:

[0026] and individual steps within the multistep process for forminglactacystin, clasto-lactacystin β-lactone or various analogs thereof.

[0027] A ninth aspect of the present invention relates to individualintermediates, such as compounds of Formulae XVII, XVIII and XIX:

[0028] where X is a halogen, preferably Cl, Br or I, as well asindividual steps within the multistep process for forming substitutedoxazolines of Formula I.

[0029] Other features or advantages of the present invention will beapparent from the following detailed description, and also from theappending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1. depicts a graph showing the effect of compound 3b,administered i.v., on infarct volume in rats (n=6-8).

[0031]FIG. 2. depicts a graph showing the effect of compound 3b,administered i.v. on neurological score in rats (n=6-8).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present invention relates to an improved multi-step synthesisof lactacystin, clasto-lactacystin β-lactone, and analogs thereof, thatproceeds in fewer steps and in much greater overall yield than synthesesdescribed in the prior art. A number of individual process steps andchemical intermediates distinguish this synthetic pathway from pathwaysdescribed in the prior art. For example, this synthetic pathway reliesupon a novel stereospecific synthesis of an oxazoline intermediate, anda unique stereoselective addition of a formyl amide to the oxazoline.

[0033] The invention is also directed to novel analogs of Formulae VIand VII that possess unexpected biological activity. Lactacystin,clasto-lactacystin β-lactone, and analogs thereof possess biologicalactivity as inhibitors of the proteasome. They can be used to treatconditions mediated directly by the function of the proteasome, such asmuscle wasting, or mediated indirectly via proteins which are processedby the proteasome, such as the transcription factor NF-κB. The presentinvention is also directed to methods of inhibiting proteasome functionor treating a condition that is mediated directly or indirectly by thefunction of the proteasome, by administering a compound of Formula VI orVII that possesses unexpectedly high activity in inhibiting theproteasome. In a preferred aspect of the invention, a pharmaceuticalcomposition that includes a compound of Formula VI or Formula VII isadministered to treat ischemic or reperfusion injury. For example, in apreferred embodiment said compounds can be used to treat, prevent orameliorate neuronal loss following stroke.

[0034] Synthetic Processes

[0035] A first aspect of the present invention relates to processes forforming lactacystin and analogs thereof having Formula VI andclasto-lactacystin β-lactone and analogs thereof having Formula VII:

[0036] wherein

[0037] R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl,aralkyl, where the ring portion of any of said aryl, aralkyl, or alkarylcan be optionally substituted;

[0038] R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy,alkoxyalkyl, or amido, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; and

[0039] R⁷ is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl,aryl, aralkyl or alkaryl can be optionally substituted.

[0040] The processes for forming these compounds rely upon formation ofa common carboxylic acid intermediate of Formula V:

[0041] where R¹ and R² are as defined above for Formulae VI and VII.These steps include:

[0042] (a) deprotonating a substituted aryl or heteroaryl oxazoline ofFormula I:

[0043] where R¹ is as defined above, and R³ is alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkaryl, any of which can be optionally substituted;and

[0044] R⁴ is aryl or heteroaryl, either of which may be optionallysubstituted; by treating said substituted aryl or heteroaryl oxazolinewith a strong base to form an enolate;

[0045] (b) transmetallating said enolate with a metal selected from thegroup consisting of titanium, aluminum, tin, zinc, magnesium and boron,and thereafter treating with a formyl amide of Formula XIV:

[0046] where R² is as defined above for Formulae VI and VII, and R⁵ andR⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ when takentogether with the nitrogen atom to which they are attached form a 5- to7-membered heterocyclic ring, which may be optionally substituted, andwhich optionally may include an additional oxygen or nitrogen atom, toform an adduct of Formula II:

[0047] where R¹ through R⁶ are as defined above;

[0048] c) catalytically hydrogenating said adduct of Formula II to forma γ-lactam of Formula IV:

[0049] where R¹, R² and R³ are as defined above;

[0050] d) saponifying said γ-lactam of Formula IV to form a lactamcarboxylic acid of Formula V:

[0051] where R¹ and R² are as defined above.

[0052] The carboxylic acid intermediate of Formula V can be cyclized bytreatment with a cyclizing reagent to form aclasto-lactacystin-β-lactone or analog thereof of Formula VII, which canbe optionally further reacted with a thiol (R⁷SH), such asN-acetylcysteine, to form lactacystin or an analog thereof havingFormula VI.

[0053] Alternatively, the carboxylic acid intermediate of Formula V canbe directly coupled to a thiol (R⁷SH), such as N-acetylcysteine, to formlactacystin or an analog thereof having Formula VI.

[0054] A second aspect of the present invention relates to the formationof enantiomerically-enriched formyl amides of Formula XIV:

[0055] wherein R², R⁵ and R⁶ are as defined above, said methodcomprising:

[0056] (a) deprotonating a compound of Formula VIII:

[0057] where R⁸ is isopropyl or benzyl, and thereafter acylating theresultant anion with R²CH₂COCl to form an acyloxazolidinone of FormulaIX:

[0058] where R² and R⁸ are as defined above;

[0059] (b) stereoselectively reacting the acyloxazolidinone of FormulaIX with benzyloxymethyl chloride to form a protected alcohol of FormulaX:

[0060] where R² and R⁸ are as defined above;

[0061] (c) hydrolyzing the protected alcohol of Formula X to form acarboxylic acid of Formula XI:

[0062] where R² is as defined above;

[0063] (d) coupling said acid of Formula XI with an amine R⁵R⁶NH₂ toprovide an amide of Formula XII:

[0064] where R², R⁵ and R⁶ are as defined above;

[0065] (e) catalytically hydrogenating, the amide of Formula XII to forman alcohol of Formula XIII:

[0066] where R², R⁵ and R⁶ are as defined above; and

[0067] (f) oxidizing the resultant alcohol of Formula XIII to give aformyl amide of Formula XIV.

[0068] A third aspect of the invention relates to a process for forminga tri-substituted cis-oxazoline compound of Formula Ia:

[0069] wherein

[0070] R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl,aralkyl, where the ring portion of any of said aryl, aralkyl, or alkarylcan be optionally substituted;

[0071] R³ is alkyl, cycloalkyl, aryl, alkaryl, any of which can beoptionally substituted; and

[0072] R⁴ is aryl or heteroaryl, either of which may be optionallysubstituted; said method comprising:

[0073] (a) asymmetrically dihydroxylating an alkene intermediate ofFormula XV:

[0074] to form an optically active diol of Formula XVIa:

[0075] (b) reacting said optically active diol of Formula XVIa with anorthoester derived from an aromatic carboxylic acid under acid catalysis(Lewis or Brönsted acid) to give a mixed orthoester, and thereafterreacting the resulting mixed orthoester intermediate with a reagentselected from the group consisting of lower alkanoyl halides, hydrohalicacids (HX, where X is a halogen), acid chlorides, and halogen-containingLewis acids (for example BBr₃, SnCl₄, Ti(OR)₂Cl₂, Ti(OR)₃Cl, Me₃SiX,where X is a halogen, and the like) in the presence of a base to form aderivative of Formula XVIIa:

[0076] wherein X is a halogen, preferably Cl, Br or I;

[0077] (c) reacting said derivative of Formula XVIIa with an alkalimetal azide to form an azide of Formula XVIIIa:

[0078] (d) catalytically hydrogenating said azide to form a compound ofFormula XIXa:

[0079] (e) subjecting the compound of Formula XIXa to ring closingconditions to form said substituted aryl- or heteroaryloxazoline ofFormula I with inversion of configuration at the oxygen-substitutedcarbon to produce a cis-oxazoline of Formula Ia; wherein for each ofFormulae XV, XVIa, XVIIa, XVIIIa and XIXa, R¹, R³ and R⁴ are as definedabove for Formula I.

[0080] Alternatively, the third aspect of the invention relates to aprocess for forming a tri-substituted trans-oxazoline compound ofFormula Ib comprising:

[0081] (a) asymmetrically dihydroxylating an alkene intermediate ofFormula XV:

[0082] to form an optically active diol of Formula XVIb:

[0083] (b) reacting said optically active diol of Formula XVIb with anorthoester derived from an aromatic carboxylic acid under acid catalysis(Lewis or Brönsted acid) to give a mixed orthoester, and thereafterreacting the resulting mixed orthoester intermediate with a reagentselected from the group consisting of lower alkanoyl halides, hydrohalicacids (HX, where X is halogen), acid chlorides, and halogen-containingLewis acids (for examples, BBr₃, SnCl₄, Ti(OR)₂Cl₂, Ti(OR)₃Cl, Me₃SiX,where X is a halogen, and the like) in the presence of a base to form aderivative of Formula XVIIb:

[0084] wherein X is a halogen, preferably Cl, Br, or I;

[0085] (c) reacting said derivative of Formula XVIIb with an alkalimetal azide to form an azide of Formula XVIIIb:

[0086] (d) catalytically hydrogenating said azide to form a compound ofFormula XIXb:

[0087] (e) subjecting the compound of Formula XIXb to ring closingconditions to form said substituted aryl- or heteroaryloxazoline ofFormula Ib, wherein the ring closure reaction proceeds with retention ofconfiguration at the oxygen-substituted carbon to produce atrans-oxazoline of Formula Ib; wherein for each of Formulae XV, XVI,XVII, XVIII and XIX, R¹, R³, and R⁴ are as defined above for Formula I.

[0088] With respect to the processes described above, the followingpreferred values are applicable:

[0089] Preferred values of R¹ are C₁₋₁₂alkyl, especially C₁₋₈alkyl,C₃₋₈cycloalkyl, especially C₃₋₆cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₆₋₁₄aryl, especially C₆₋₁₀aryl, C₆₋₁₀ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl, where the ring portion of any of said aryl, aralkyl,or alkaryl can be optionally substituted. Substituents that can beoptionally present on the aryl ring of an R¹ moiety include one or more,preferably one or two, of hydroxy, nitro, trifluoromethyl, halogen,C₁₋₆alkyl, C₆₋₁₀aryl, C₁₋₆alkoxy, C₁₋₆aminoalkyl, C₁₋₆aminoalkoxy,amino, C₂₋₆alkoxycarbonyl, carboxy, C₁₋₆hydroxyalkyl, C₂₋₆hydroxyalkoxy,C₁₋₆alkylsulfonyl, C₆₋₁₀arylsulfonyl, C₁₋₆alkylsulfinyl,C₁₋₆alkylsulfonamido, C₆₋₁₀arylsulfonamido, C₆₋₁₀ar(C₁₋₆)alkylsulfonamido, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₆₋₁₀aryl, C₆₋₁₀aryl(C₁₋₆)alkyl, C₁₋₆alkylcarbonyl, C₂₋₆ carboxyalkyl, cyano, andtrifluoromethoxy.

[0090] R¹ is more preferably one of C₁₋₈alkyl such as ethyl, propyl orisopropyl; cycloalkyl, such as cyclohexyl; or C₆₋₁₀aryl, such as phenyl.Most preferred is isopropyl.

[0091] Preferred values of R² are C₁₋₈alkyl, C₃₋₈cycloalkyl, especiallyC₃₋₆cycloalkyl, C₁₋₈alkoxy, C₂₋₈alkenyl, C₂₋₈alkynyl C₆₋₁₄aryl,especially C₆₋₁₀aryl, C₆₋₁₀ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, wherethe ring portion of any of said aryl, aralkyl, or alkaryl can beoptionally substituted with any of the substituents as described for R¹above.

[0092] R² is more preferably C₁₋₄alkyl, such as methyl, ethyl, propyl,or butyl; or C₁₋₄alkoxy, such as methoxy, or ethoxy. Most preferred aremethyl, ethyl and propyl, and butyl.

[0093] With respect to R³, a variety of ester functionalities can beemployed at this position. Preferred values are C₁₋₈alkyl,C₃₋₈cycloalkyl, especially C₄₋₇cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₆₋₁₄aryl, especially C₆₋₁₀aryl, C₆₋₁₀ar(C₁₋₆) alkyl orC₁₋₆alk(C₆₋₁₀)aryl, any of which can be optionally substituted.Substituents that can be optionally present on R³ include one or more,preferably one or two, of the substituents as described for R¹ above.

[0094] R³ is more preferably C₁₋₄alkyl, C₆₋₁₀aryl or C₆₋₁₀ar(C₁₋₆)alkyl. Most preferred are methyl, ethyl, tert-butyl and benzyl.

[0095] R⁴ is preferably C₆₋₁₀ aryl, preferably phenyl, or a heteroarylgroup selected from the group consisting of thienyl, benzo[b]thienyl,furyl, pyranyl, isobenzofuranyl, benzoxazolyl, 2H-pyrrolyl, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,4H-quinolizinyl, isoquinolyl, quinolyl, ortriazolyl. The phenyl orheteroaryl group can be optionally substituted by one or two of thesubstituents as described for R¹ above. Most preferred are phenyl, andphenyl substituted by halogen, C₁₋₆alkyl, C₁₋₆alkoxy, carboxy, amino,C₁₋₆alkylamino and/or di(C₁₋₆)alkylamino.

[0096] R⁵ and R⁶ are independently one of alkyl, aralkyl or alkaryl; orR⁵ and R⁶ when taken together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocyclic ring, which can beoptionally substituted, and which optionally can include an additionaloxygen or nitrogen atom. Optional substituents are those listed abovefor R¹.

[0097] R⁵ and R⁶ are preferably C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl or together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocycle which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom. Most preferred values for NR⁵R⁶ are dimethylamino,diethylamino, pyrrolidino, piperidino, morpholino, oxazolidinone, andoxazolidinone substituted by halogen, C₁₋₆alkyl, C₆₋₁₀ ar(C₁₋₆)alkyl,C₁₋₆alkoxy, carboxy, and/or amino.

[0098] R⁷ is preferably C₁₋₈alkyl, C₃₋₈cycloalkyl, C₆₋₁₀aryl,C₆₋₁₀ar(C₁₋₆)alkyl, C₁₋₆alk(C₆₋₁₀)aryl, any of which can be optionallysubstituted. Substituents that can be optionally present on either orboth of the ring or chain portions of R⁷ include one or more, preferablyone or two, of the substituents as described for R¹ above. Preferably,R⁷ together with the sulfur atom to which it is attached is cysteine ora derivative of cysteine such as N-acetyl cysteine, glutathione, and thelike.

[0099] Scheme 1 is a general scheme for forming lactacystin andclasto-lactacystin-β-lactone analogs from substituted oxazoline startingmaterials.

[0100] The starting oxazoline I, which may be of either the cis (Ia) ortrans (Ib) configuration, is deprotonated with a strong base to form theenolate. Examples of bases suitable for use in this reaction are organicbases, including hindered amide bases such as lithium diisopropylamide(LDA), lithium tetramethylpiperidide (LiTMP), lithium, sodium orpotassium hexamethyldisilazide (LiHMDS, NaHMDS, KHMDS), or the like; orhindered alkyllithium reagents, such as sec-butyllithium,tert-butyllithium, or the like. The reaction is preferably conducted atreduced temperature in an ethereal solvent, such as diethyl ether,tetrahydrofuran (THF), or dimethoxyethane (DME). Reaction temperaturespreferably range from about −100° C. to about −30° C., more preferablyfrom −85° C. to −50° C., and most preferably from −85° C. to −75° C. Thereaction temperature is important in determining the stereochemicaloutcome of the subsequent addition to the aldehyde, with lowertemperatures providing better selectivity.

[0101] The deprotonation step is followed by transmetallating saidenolate with a metal selected from the group consisting of titanium,aluminum, tin, zinc, magnesium and boron. Preferred reagents for thisstep include titanium or aluminum Lewis acids, for example Me₂AlCl or(i-PrO)₃TiCl or a mixture of the two. Preferably, between one and threemolar equivalents of the Lewis acid are used, more preferably betweentwo and three equivalents, and most preferably about 2.2-2.3equivalents. Subsequent treatment of the enolate with a formyl amide(XIV) affords the adduct II. Excess aldehyde is washed away with sodiumbisulfite solution, and the crude material is carried forward to thenext step without further purification. The use of 2.2-2.3 equivalentsof Me₂AlCl results in selective formation of the (6S)-product(lactacystin numbering), in a ratio generally better than about 10:1,whereas the use of 1 equivalent of Me₂AlCl results in selectiveformation of the (6R)-product, in a ratio of about 5:1.

[0102] Catalytic hydrogenolysis of the adduct II, as a mixture of (6S)-and (6R)-epimers, affords the desired γ-lactam (IV), sometimes as amixture with the aminodiol III:

[0103] Useful catalysts for this reaction include palladium black,palladium on activated carbon, palladium hydroxide on carbon, or thelike. Organic solvents suitable for use in this reaction include loweralkanols such as methanol, ethanol, or isopropanol, lower alkanoatessuch as ethyl acetate, lower alkanoic acids such as acetic acid, ormixtures thereof. The reaction is conducted under an atmosphere ofhydrogen, at pressures ranging from about 15 to about 100 p.s.i., morepreferably from about 30 to about 50 p.s.i. Alternatively, transferhydrogenation procedures (R. A. W. Johnstone et al., Chem. Rev. 85:129(1985)) may be used, in which the adduct II is treated at atmosphericpressure with a catalyst and a hydrogen donor.

[0104] Upon heating of the crude product mixture, the aminodiol III isconverted to the γ-lactam IV, which can then be isolated inapproximately 60-75% overall yield from II. The heating step isconveniently carried out by first filtering off the catalyst used in thehydrogenation step and then heating the filtrate to reflux. When noaminodiol III is present in the crude product mixture, the heating stepis omitted. Ester saponification, followed by cyclization, affords theβ-lactone VII in 40-90% yield, and generally in greater than 60% yield.Cyclization can be effected with coupling reagents known in the art,including aryl sulfonyl chlorides,benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent), O-(1 H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TBTU), alkyl, aryl or alkenyl chloroformates, and thelike. Isopropenyl chloroformate is a preferred reagent for this step,since all byproducts are volatile and chromatographic purification ofthe product is not necessary.

[0105] clasto-Lactacystin β-lactone can be converted to lactacystin bytreatment of the β-lactone with N-acetylcysteine according to thereported procedure (Corey et al., Tetrahedron Lett. 34:6977 (1993)).Reactions of the β-lactone VII with other thiols proceed analogously.Alternatively, lactacystin analogs are prepared by coupling thecarboxylic acid intermediate V with a thiol to form the correspondingthiolester VI. The method of this invention is therefore useful forsynthesis of both lactacystin and clasto-lactacystin β-lactone, as wellas analogs thereof.

[0106] The enantiomerically-enriched formyl amides XIV employed in thealdol reaction are new. They can be prepared according to arepresentative reaction sequence such as that depicted in Scheme 2. Forpurposes of the present invention, the term “enantiomerically-enriched”means that one enantiomer is present in excess relative to the other;that is, one enantiomer represents greater than 50% of the mixture. Theterm “stereoselective” is used to mean that a synthesis or reaction stepproduces one enantiomer or diastereomer in excess relative to the otherenantiomer or to other diastereomer(s).

[0107] Acylation of the anion of (S)-(−)-4-benzyl-2-oxazolidinone(VIIIa) or (S)-(−)-4-isopropyl-2-oxazolidinone (VIIIb) (where R⁸ isbenzyl or isopropyl) affords the acyloxazolidinone IX in greater than80% yield. Subsequent stereoselective benzyloxymethylation (Evans etal., J. Am. Chem. Soc. 112:8215 (1990)) gives the protected alcohol X ingreater than 80% yield, provided that the benzyl chloromethyl ether isfreshly prepared (Connor et al., Organic Syntheses 52:16 (1974)).Peroxide mediated hydrolysis affords the acid XI, which is coupled withan amine to provide the amide XII, generally in greater than 50% overallyield. Benzyl group hydrogenolysis, followed by oxidation of theresultant alcohol (XIII) then affords the formyl amide XIV in 80-85%yield. Pearlmans catalyst (Pd(OH)₂) is preferably used for thehydrogenolysis step. The final oxidation step is best accomplished withthe periodinane reported by Dess and Martin, J. Org. Chem. 48:4156(1983) or with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) freeradical, and buffered hypochlorite in the presence of bromide ion (J.Org. Chem. 50:4888 (1985); Org. Synth. Coil. 8:367 (1993)). Other mildoxidants such as tetrapropyl-ammonium perruthenate (TPAP) can also beused. The formyl amide XIV can be shown to be enantiomerically pure byreducing the aldehyde with sodium borohydride and converting theresultant alcohol to the corresponding Mosher ester usingR-(+)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (Dale et al.,J. Org. Chem. 34:2543 (1969)). ¹H NMR analysis at 300 MHz reveals asingle diastereomer. The aldehydes prepared according to Scheme 2 areconfigurationally stable, showing no signs of enantiomeric deteriorationafter one week, when stored at 0° C. The aldehyde is alsoconfigurationally stable under the conditions of the aldol reaction, andthe the adduct II is formed without epimerization of the substituent R²at C(7).

[0108] The synthetic methods will work with any substituent at R¹ thatis stable to strong base and to hydrogenation. Isopropyl is thepreferred substituent for good proteasome inhibiting activity of thefinal product.

[0109] The invention also relates to a new route to form the oxazolinestarting material I. The overall synthesis includes five steps (Scheme3) and affords the cis-substituted oxazoline Ia, which is thereafteremployed in the method described above. The first step depicted inScheme 3 is Sharpless asymmetric dihydroxylation (Sharpless et al., J.Org. Chem. 57:2768 (1992); Kolb et al., Chem. Rev. 94:2483 (1994); Shaoand Goodman, J. Org. Chem. 61:2582(1996)) of the alkene XV. If notcommercially available, the alkene XV is prepared by Wittig condensationbetween the aldehyde and carboethoxymethylene triphenylphosphorane (Haleet al., Tetrahedron 50:9181 (1994)). Other olefination procedures arealso known in the art. The dihydroxylation reaction is preferablyconducted with AD-mix-β (Aldrich Chemical Co.) in the presence ofmethane sulfonamide and stereoselectively affords the diol XVIa, aspredicted by the Sharpless face-selection rule. On a large scale, thedihydroxylation reaction is preferably conducted usingN-methylmorpholine-N-oxide (NMO) as the reoxidant in place of K₃Fe(CN)₆present in AD-mix-β. Although proceeding with somewhat lowerenantioselectivity, this procedure allows more concentrated reactionmixtures and greatly simplifies the workup. The enantiomeric purity ofthe product can be enhanced by recrystallization.

[0110] In the next step, the diol XVIa is treated with an orthoesterunder Lewis or Brönsted acid catalysis to give a mixed orthoester, whichis converted in situ to the haloester XVIIa by treatment with an acylhalide (Haddad et al., Tetrahedron Lett. 37:4525 (1996)). Although acylhalides, especially acetyl halides are preferred for this reaction,other acid halides such as HCl, HBr, HI, Me₃SiCl, Me₃SiI, Me₃SiBr andthe like may be used. Halogen-containing Lewis acids of the formulaML_(n)X, such as BBr₃, SnCl₄, Ti(OR)₂Cl₂, Ti(OR)₃Cl, and the like canalso be used. In the previous formula, M is a metal selected from thegroup consisting of B, Ti, Sn, Al, Zn, and Mg; L is any suitable ligandfor the metal, preferably an alkoxide or halogen group; n is an integerthat results in a stable complex, and X is a halogen. Preferably acetylbromide is used to produce the haloester XVIIa. Preferably theorthoester employed in this reaction is derived from an aromatic orheteroaromatic carboxylic acid. More preferably, the orthoester isderived from benzoic acid, e.g., trimethyl orthobenzoate. The use ofboron trifluoride etherate as the Lewis acid catalyst in the formationof the mixed orthoester is preferred, but other acids, such as HBr,SnCl₄, TiCl₄, BBr₃, and the like, can also be used.

[0111] After workup, the crude halide XVIIa is converted to the azideXVIIIa by treatment with an alkali metal azide in a polar aproticorganic solvent, such as dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF). Catalytic hydrogenation of the azide XVIIIa over apalladium catalyst in ethyl acetate proceeds with concomitant migrationof the aroyl group (Wang et al., J. Org. Chem. 59:5014 (1994)) to affordthe hydroxyamide XIXa.

[0112] Treatment of XIXa with thionyl chloride in methylene chlorideeffects ring closure with inversion of configuration at thehydroxyl-substituted carbon atom to produce the cis-substitutedoxazoline starting material Ia. Other reagents suitable for use in thisreaction include sulfuryl chloride, phosphorous trichloride, phosphorousoxychloride, and (methoxycarbonylsulfamoyl)-triethylammonium hydroxide,inner salt (Burgess reagent). Treatment of XIXa under Mitsunobuconditions (Mitsunobu, Synthesis:1 (1981) will also effect a ringclosure. The oxazoline ring oxygen atom is destined to become theC(9)-hydroxyl group in the final products VI and VII. Underequilibrating conditions (sodium methoxide, methanol), the cis-oxazoline(Ia) is converted to the trans-oxazoline (Ib) by inversion ofconfiguration of the ester substituent, with the configuration of the R¹substituent remaining fixed. The cis- and trans-oxazolines can both beused in the method depicted in Scheme 1, with equivalent results.

[0113] In an alternative route to form the oxazoline starting materialI, p-toluenesulfonic acid (p-TsOH) is used to effect ring closure(Scheme 4). In this case, ring closure proceeds with retention ofconfiguration at the hydroxyl-substituted carbon atom to afford thetrans-oxazoline (Ib). In order to obtain the proper stereochemistry atC(9) of the final product, the chiral ligand employed in thedihydroxylation reaction must be selected so as to provide the oppositeface selectivity from that depicted in Scheme 3. For example, AD-mix-αis used in place of AD-mix-β. All other steps in the sequence proceedanalogously to those described for the synthesis of the cis-oxazolineIa.

[0114] Compounds

[0115] Many of the compounds described above are novel compounds; thenovel compounds are also claimed.

[0116] Fourth, fifth and sixth aspects of the invention relate tolactacystin analogs that can be made by the synthetic routes describedherein; to pharmaceutical compositions including such compounds; and tomethods of treating a subject having a condition mediated by proteinsprocessed by the proteasome by administering to a subject an effectiveamount of a pharmaceutical composition disclosed herein. These methodsinclude treatments for Alzheimers disease, cachexia, cancer,inflammation (e.g., inflammatory responses associated with allergies,bone marrow or solid organ transplantation, or disease states, includingbut not limited to arthritis, multiple sclerosis, inflammatory boweldisease and parasitic diseases such as malaria), psoriasis, restenosis,stroke, and myocardial infarction.

[0117] The compounds of Formulae VI and VII disclosed herein are highlyselective for the proteasome, and do not inhibit other proteases such astrypsin, α-chymotrypsin, calpain I, calpain II, papain, and cathepsin B.

[0118] As disclosed by Fenteany et al. (WO 96/32105), herebyincorporated by reference in its entirety, lactacystin,clasto-lactacystin β-lactone, and analogs thereof possess biologicalactivity as inhibitors of the proteasome. They can be used to treatconditions mediated directly by the function of the proteasome, such asmuscle wasting, or mediated indirectly via proteins which are processedby the proteasome, such as the transcription factor NF-κB. The compoundsprepared by the methods of this invention can also be used to determinewhether a cellular, developmental, or physiological process or output isregulated by the proteolytic activity of the proteasome.

[0119] Those compounds that possess unexpected proteasomefunction-inhibiting activity are compounds of Formulae VI and VII:

[0120] or a salt thereof, wherein:

[0121] R¹ is C₁₋₁₂alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl;

[0122] R² is C₂₋₆alkyl; and

[0123] R⁷ is C₁₋₈ alkyl, C₃₋₈cycloalkyl, C₆₋₁₀aryl, C₆₋₁₀ar(C₁₋₆)alkyl,C₁₋₆alk(C₆₋₁₀)aryl, any of which can be optionally substituted.Substituents that can be optionally present on either or both of thering or chain portions of R⁷ include one or more, preferably one or two,of the substituents as described for R¹ above.

[0124] Preferred compounds are those where R¹ is C₁₋₄ alkyl, morepreferably isopropyl. R² is preferably ethyl, n-propyl, n-butyl orisobutyl. Preferably, R⁷ together with the sulfur atom to which it isattached is cysteine or a derivative of cysteine such as N-acetylcysteine, glutathione, and the like.

[0125] A seventh aspect of the present invention is directed toenantiomerically-enriched formyl amides of Formula XIV:

[0126] or salts thereof, wherein

[0127] R² is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl; and

[0128] R⁵ and R⁶ are independently C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl, or together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocycle which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom.

[0129] Preferred compounds are those where R² is C₂₋₆alkyl.

[0130] An eighth aspect of the present invention is directed tocompounds of Formulae II and III:

[0131] or salts thereof wherein

[0132] R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl,aralkyl, where the ring portion of any of said aryl, aralkyl, or alkarylcan be optionally substituted;

[0133] R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy,alkoxyalkyl, or amido, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted;

[0134] R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any ofwhich can be optionally substituted;

[0135] R⁴ is optionally substituted aryl or optionally substitutedheteroaryl; and

[0136] R⁵ and R⁶ are independently one of alkyl or alkaryl; or R¹ and R⁶when taken together with the nitrogen atom to which they are attachedform a 5- to 7-membered heterocyclic ring, which can be optionallysubstituted, and which optionally include an additional oxygen ornitrogen atom. Most preferred values for NR⁵R⁶ are dimethylamino,diethylamino, pyrrolidino, piperidino, morpholino, oxazolidinone, andoxazolidinone substituted by halogen, C₁₋₆alkyl, C₆₋₁₀ ar(C₁₋₆alkyl,C₁₋₆alkoxy, carboxy, and/or amino.

[0137] Preferred compounds of Formulae II and III are those wherein:

[0138] R¹ is C₁₋₁₂alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, where the ringportion of any of said aryl, aralkyl, or alkaryl can be optionallysubstituted;

[0139] R² is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, where the ringportion of any of said aryl, aralkyl, or alkaryl can be optionallysubstituted;

[0140] R³ is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₆₋₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, any of which can beoptionally substituted;

[0141] R⁴ is optionally substituted C₆₋₁₀aryl, or an optionallysubstituted heteroaryl group selected from the group consisting ofthienyl, benzo[β]thienyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, ortriazolyl; and

[0142] R⁵ and R⁶ are independently C₁₋₆alkyl, C₆₋₁₀ ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl, or together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocycle which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom. Most preferred values for NR⁵R⁶ are dimethylamino,diethylamino, pyrrolidino, piperidino, morpholino, oxazolidinone, andoxazolidinone substituted by halogen, C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkyl,C₁₋₆ alkoxy, carboxy, and/or amino.

[0143] A ninth aspect of the present invention is directed to compoundsof Formulae XVIIa, XVIIb, XVIIIa, XVIIIb, XIXa or XIXb:

[0144] or salts thereof, wherein

[0145] R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl,aralkyl, where the ring portion of any of said aryl, aralkyl, or alkarylcan be optionally substituted;

[0146] R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any ofwhich can be optionally substituted; and

[0147] R⁴ is optionally substituted aryl or optionally substitutedheteroaryl.

[0148] Preferred compounds of Formulae XVII, XVIII or XIX are thosewherein %o R¹ is C₁₋₁₂alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynylC₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, where the ringportion of any of said aryl, aralkyl, or alkaryl can be optionallysubstituted;

[0149] R³ is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, any of which canbe optionally substituted; and

[0150] R⁴ is optionally substituted C₆₋₁₀aryl, or an optionallysubstituted heteroaryl group selected from the group consisting ofthienyl, benzo[b]thienyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, ortriazolyl.

[0151] Definitions

[0152] The term “alkyl” as employed herein includes both straight andbranched chain radicals of up to 12 carbons, preferably 1-8 carbons,such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl,pentyl, hexyl, isohexyl, 1-ethylpropyl, heptyl, 4,4-dimethylpentyl,octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl.

[0153] The term “substituted alkyl” as employed herein, includes alkylgroups as defined above that have one, two or three halo, hydroxy,nitro, trifluoromethyl, halogen, C₁₋₆alkyl, C₆₋₁₀aryl, C₁₋₆alkoxy,C₁₋₆aminoalkyl, C₁₋₆aminoalkoxy, amino, C₂₋₆alkoxycarbonyl, carboxy,C₁₋₆hydroxyalkyl, C₂₋₆hydroxyalkoxy, C₁₋₆ alkylsulfonyl,C₆₋₁₀arylsulfonyl, C₁₋₆alkylsulfinyl, C₁₋₆alkylsulfonamido, C₆₋₁₀arylsulfonamido, C₆₋₁₀ar(C₁₋₆)alkylsulfonamido, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₆₋₁₀ aryl, C₆₋₁₀aryl(C₁₋₆)alkyl, C₁₋₆alkylcarbonyl,C₂₋₆carboxyalkyl, cyano, and trifluoromethoxy and/or carboxysubstituents.

[0154] The term “cycloalkyl” as employed herein includes saturatedcyclic hydrocarbon groups containing 3 to 12 carbons, preferably 3 to 8carbons, which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl, any of whichgroups may be substituted with substituents such as halogen, C₁₋₆alkyl,C₁₋₆alkoxy and/or hydroxy group.

[0155] The term “heteroaryl” as employed herein refers to groups having5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; 6, 10 or 14 πelectrons shared in a cyclic array; and containing carbon atoms and 1, 2or 3 oxygen, nitrogen or sulfur heteroatoms (where examples ofheteroaryl groups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl,thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl,xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl,3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl,quinolyl, phthalazinyl, naphthyridinyl, tetrazolyl, quinazolinyl,cinnolinyl, pteridinyl, 4αH-carbazolyl, carbazolyl, β-carbolinyl,phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl,isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl and phenoxazinylgroups).

[0156] The term “aryl” as employed herein by itself or as part ofanother group refers to monocyclic or bicyclic aromatic groupscontaining from 6 to 12 carbons in the ring portion, preferably 6-10carbons in the ring portion, such as phenyl, naphthyl ortetrahydronaphthyl.

[0157] The term “aralkyl” or “arylalkyl” as employed herein by itself oras part of another group refers to C₁₋₆alkyl groups as discussed abovehaving an aryl substituent, such as benzyl, phenylethyl or2-naphthylmethyl.

[0158] The term “alkaryl” or “alkylaryl” as employed herein by itself oras part of another group refers to an aryl group as discussed abovehaving a C₁₋₆ alkyl substituent, such as toluyl, ethylphenyl, ormethylnaphthyl.

[0159] The term “optionally substituted” when used with respect to aryl,aralkyl, alkaryl or 5-, 6-, 9- or 10-membered heteroaryl groups meansthat the ring portion of said groups can be optionally substituted byone or two substituents independently selected from C₁₋₆alkyl,C₃₋₈cycloalkyl, C₁₋₆alkyl(C₃₋₈)cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,cyano, amino, C₁₋₆alkylamino, di(C₁₋₆)alkylamino, benzylamino,dibenzylamino, nitro, carboxy, carbo(C₁₋₆)alkoxy, trifluoromethyl,halogen, C₁₋₆alkoxy, C₆₋₁₀aryl, C₆₋₁₀aryl(C₁₋₆)alkyl,C₆₋₁₀aryl(C₁₋₆)alkoxy, hydroxy, C₁₋₆alkylthio, C₁₋₆alkylsulfinyl,C₁₋₆alkylsulfonyl, C₆₋₁₀arylthio, C₆₋₁₀arylsulfinyl, C₆₋₁₀arylsulfonyl,C₆₋₁₀aryl, C₁₋₆alkyl(C₆₋₁₀)aryl, and halo(C₆₋₁₀)aryl.

[0160] The term “alkoxy” refers to the above alkyl groups linked tooxygen.

[0161] The term “halogen” or “halo” as employed herein by itself or aspart of another group refers to chlorine, bromine, fluorine or iodine.

[0162] The term “amido” as employed herein refers to formylamino,alkylcarbonylamino or arylcarbonylamino.

[0163] Uses

[0164] Pharmacological data for clasto-lactacystin β-lactone analogsprepared by the methods of this invention are provided in Table 1. Thesecompounds are all irreversible inactivators of the 20S proteasome,acylating the N-terminal threonine residue of the X/MB1 subunit. Thevalue K_(obs)/[I] is a measure of the rate of enzyme inactivation.Several compounds show improved activity, i.e., more rapid rates ofinactivation, when compared to clasto-lactacystin β-lactone itself(2).The compound that is most potent in the enzyme assay is the 7-methoxyderivative 3f. However, when assayed in cell culture, 3f is less potentthan 2.

[0165] The lactone ring is subject to nucleophilic attack not only bythe threonine residue of the proteasome X/MB1 subunit, but also bywater. Hydrolysis results in formation of the hydroxy acid V, which isnot active as an inhibitor of the proteasome. Relative potency in cellculture is a composite of many factors, including enzyme potency, cellpenetration, and hydrolysis rate. Although more potent than 2 againstthe enzyme, 3f is also more rapidly hydrolyzed, resulting in much weakeractivity in cell culture. By contrast, the analogs 3a-3d showunexpectedly improved potency not only in the enzyme assay, but also incell culture.

[0166] The disclosed compounds are used to treat conditions mediateddirectly by the proteolytic function of the proteasome such as musclewasting, or mediated indirectly via proteins which are processed by theproteasome such as NF-κB. The proteasome participates in the rapidelimination and post-translational processing of proteins involved incellular regulation (e.g., cell cycle, gene transcription, and metabolicpathways), intercellular communication, and the immune response (e.g.,antigen presentation). Specific examples include β-amyloid protein andregulatory proteins such as cyclins and transcription factor NF-κB.Treating as used herein includes reversing, reducing, or arresting thesymptoms, clinical signs, and underlying pathology of a condition in amanner to improve or stabilize the subject's condition.

[0167] Other embodiments of the invention relate to cachexia andmuscle-wasting diseases. The proteasome degrades many proteins inmaturing reticulocytes and growing fibroblasts. In cells deprived ofinsulin or serum, the rate of proteolysis nearly doubles.

[0168] Inhibiting the proteasome reduces proteolysis, thereby reducingboth muscle protein loss and the nitrogenous load on kidneys or liver.Proteasome inhibitors are useful for treating conditions such as cancer,chronic infectious diseases, fever, muscle disuse (atrophy) anddenervation, nerve injury, fasting, renal failure associated withacidosis, and hepatic failure. See, e.g., Goldberg, U.S. Pat. No.5,340,736 (1994).

[0169] Embodiments of the invention therefore encompass methods forreducing the rate of muscle protein degradation in a cell, and reducingthe rate of intracellular protein degradation. Each of these methodsincludes the step of contacting a cell (in vivo or in vitro, e.g., amuscle in a subject) with an effective amount of a compound (e.g.,pharmaceutical composition) of a formula disclosed herein.

[0170] Proteasome inhibitors block processing of ubiquitinated NF-κB invitro and in vivo. Proteasome inhibitors also block IκB-α degradationand NF-κB activation. (Palombella, et al.; and Traenckner, et al., EMBOJ. 13:5433-5441 (1994)). One embodiment of the invention is a method forinhibiting IκB-α degradation, including contacting the cell with acompound of a formula described herein. A further embodiment is a methodfor reducing the cellular content of NF-κB in a cell, muscle, organ, orsubject, including contacting the cell, muscle, organ, or subject with acompound of a formula described herein. Additional embodiments encompassmethods for treating inflammatory responses associated with allergies,bone marrow or solid organ transplantation, or disease states, includingbut not limited to arthritis, inflammatory bowel disease, asthma, andmultiple sclerosis by administering a compound of a formula disclosedherein. A preferred embodiment of the invention is directed to treatingasthma by administering a compound of Formula VI or Formula VII, mostpreferably compound 3b.

[0171] Proteasome inhibitors are also useful for treatment of ischemicor reperfusion injury, particularly for preventing or reducing the sizeof infarct after vascular occlusion such as occurs during a stroke orheart attack, as described in Brand, U.S. patent application Ser. No.______ (ProScript Docket No. 102.603.173), filed Feb. 17, 1998, U.S.patent application Ser. No. 08/988,339, filed Dec. 3, 1997, and U.S.patent application Ser. No. 08/801,936, filed Feb. 15, 1998. Proteasomeinhibitors also block proteasome-dependent transformation of protazoanparasites (Gonzalez et al., J. Exp. Med. 184:1909 (1996). Furtherembodiments of the invention therefore encompass methods for treating aninfarct or a protazoan parasitic disease by administering a compound ofa formula disclosed herein. In a preferred aspect of the invention, acompound of Formula VI or Formula VII is administered to prevent orreduce the size of the infarct after vascular occlusion. Said compoundscan be administered from about 0 to about 10 hours after the occurrenceof a stroke in order to treat or reduce neuronal loss following anischemic event. Compounds 3b is the most preferred compound in thisaspect of the invention.

[0172] Proteasome inhibitors also block degradation of cell cycleregulatory proteins, such as cyclins and cyclin-dependent kinaseinhibitors, and tumor suppressor proteins, such as p53. Otherembodiments of the invention therefore encompass methods for blockingthe cell cycle and for treating cell proliferative diseases such ascancer, psoriasis, and restenosis with a compound of a formula describedherein.

[0173] The term “inhibitor” is meant to describe a compound that blocksor reduces the activity of an enzyme (e.g., the proteasome, or the X/MB1subunit of the 20S proteasome). An inhibitor may act with competitive,uncompetitive, or noncompetitive inhibition. An inhibitor may bindreversibly or irreversibly, and therefore the term includes compoundswhich are suicide substrates of an enzyme. An inhibitor may modify oneor more sites on or near the active site of the enzyme, or it may causea conformational change elsewhere on the enzyme.

[0174] Amounts and regimens for the administration of proteasomeinhibitors and compositions of the invention can be determined readilyby those with ordinary skill in the clinical art. Generally, the dosageof the composition of the invention will vary depending uponconsiderations such as: type of composition employed; age; health;medical conditions being treated; kind of concurrent treatment, if any,frequency of treatment and the nature of the effect desired; extent oftissue damage; gender; duration of the symptoms; and, counterindications, if any, and other variables to be adjusted by theindividual physician. A desired dosage can be administered in one ormore applications to obtain the desired results. Pharmaceuticalcompositions containing the proteasome inhibitors of the invention canbe provided in unit dosage forms.

[0175] Compositions within the scope of this invention include allcompositions wherein the compounds of the present invention arecontained in an amount which is effective to achieve its intendedpurpose. While individual needs vary, determination of optimal ranges ofeffective amounts of each component is within the skill of the art.Typically, the compounds may be administered to mammals, e.g. humans,orally at a dose of 0.0025 to 50 mg/kg, or an equivalent amount of thepharmaceutically acceptable salt thereof, per day of the body weight ofthe mammal being treated for a proteosome-mediated condition such as astroke or asthma. For intramuscular injection, the dose is generallyabout one-half of the oral dose.

[0176] In the method of prevention or reduction of infarct size thecompound can be administered by intravenous injection at a dose of about0.01 to about 10 mg/kg, preferably about 0.025 to about 1 mg/kg.

[0177] The unit oral dose may comprise from about 0.01 to about 50 mg,preferably about 0.1 to about 10 mg of the compound. The unit dose maybe administered one or more times daily as one or more tablets eachcontaining from about 0.1 to about 10, conveniently about 0.25 to 50 mgof the compound or its solvates. For use in treating stroke, it ispreferred that a single dosage be administered, 0 to about 10 hourspost-event, preferably 0 to about 6 hours post-event.

[0178] The following examples are illustrative, but not limiting, of themethod and compositions of the present invention. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered and obvious to those skilled in the artare within the spirit and scope of the invention.

[0179] The preparation of formyl amides XIV according to the syntheticscheme depicted in scheme 2 as exemplified in Examples 1-6.

EXAMPLE 1 Acyl Oxazolidinones (IX)

[0180] a. Acyl oxazolidinone IXb (R²=n-Pr; R⁸=CH₂Ph): A cooled (−78° C.)solution of (S)-(−)-4-benzyl-2-oxazolidinone (4.0 g, 22.6 mmol) in 75 mLanhydrous THF was treated with a 2.5 M solution of n-BuLi in hexane (9.1mL, 22.6 mmol) over 15 min. After 5 min, neat valeryl chloride (2.95 mL,24.9 mmol) was added dropwise and the mixture was stirred for another 45min. at −78° C. The mixture was then allowed to reach room temperature,stirred for another 90 min, and then treated with 50 mL saturated NH₄Clsolution. Dichloromethane (50 mL) was then added and the organic layerwas washed with brine (2×30 mL), dried over MgSO₄ and concentrated invacuo. This afforded 5.94 g (100%) of the desired acyl oxazolidinone IXbas a clear colorless oil. ¹H NMR (300 MHz, CDCl₃) δ7.36-7.20 (m, 5H),4.71-4.64 (m, 1H), 4.23-4.14 (m, 1H), 3.40 (dd, J=13.3, 3.2 Hz, 1H),3.04-2.84 (m, 2H), 2.77 (dd, J=13.3, 9.6 Hz, 1H), 1.74-1.63 (m, 2H),1.46-1.38 (m, 2H), 0.96 (t, J=7.3 Hz, 3H).

[0181] b. Acyl oxazolidinone IXa (R²=Et; R⁸=CH₂Ph): By a procedureanalogous to that described for preparing acyl oxazolidinone IXb, thelithium anion of (S)-(−)-4-benzyl-2-oxazolidinone was treated withbutyryl chloride to provide acyl oxazolidinone IXa in 94% yield. ¹H NMR(300 MHz, CDCl₃) δ7.37-7.20 (m, 5H), 4.68 (ddd, J=13.1, 7.0, 3.4 Hz,1H), 4.23-4.13 (m, 2H), 3.30 (dd, J=13.3, 9.6 Hz, 1H), 3.02-2.82 (m,2H), 2.77 (dd, J=13.3, 9.6Hz, 1H), 1.73 (q, J=7.3 Hz, 2H), 1.01 (t,J=7.3 Hz, 3H).

[0182] c. Acyl oxazolidinone IXc (R²=n-Bu; R⁸=CH₂Ph): By a procedureanalogous to that described for preparing acyl oxazolidinone IXb, thelithium anion of (S)-(−)-4-benzyl-2-oxazolidinone was treated withhexanoyl chloride to provide acyl oxazolidinone IXc in 96% yield. ¹H NMR(300 MHz, CDCl₃) δ7.36-7.20 (m, 5H), 4.68 (m, 1H), 4.23-4.14 (m, 2H),3.30 (dd, J=13.3, 3.3 Hz, 1H), 3.02-2.83 (m, 2H), 2.76 (dd, J=13.3, 9.6Hz, 1H), 1.70 (m, 2H), 1.43-1.34 (m, 4H), 0.92 (t, J=3.3 Hz, 3H).

[0183] d. Acyl oxazolidinone IXd (R²=i-Bu; R⁸=CH₂Ph):

[0184] i. 4-Methylvaleryl chloride

[0185] 4- Methylvaleryl chloride was prepared from commerciallyavailable 4-methylvaleric acid in the following way: a cold (0° C.)solution of 4-methylvaleric acid (1.85 mL, 15.0 mmol) in 50 mL anhydrousCH₂Cl₂ containing 10 mL of DMF was treated with 1.95 μL oxalyl chloride(22.5 mmol). The mixture was then stirred for 3 h at room temperature,concentrated in vacuo and filtered to affords 1.65 g (100%) of thedesired acid chloride as a colorless liquid.

[0186] ii. Acyl oxazolidinone IXd (R²=i-Bu; R⁸=CH₂Ph):

[0187] By a procedure analogous to that described for preparing acyloxazolidinone IXb, the lithium anion of (S)-(−)-4-benzyl-2-oxazolidinonewas treated with 4-methylvaleryl chloride to provide acyl oxazolidinoneIXd in 85% yield. ¹H NMR (300 MHz, CDCl₃) δ7.37-7.20 (m, 5H), 4.70-4.63(m, 1H), 4.23-4.15 (m, 2H), 3.30 (dd, J=13.2, 3.2 Hz, 1H), 2.98-2.90 (m,2H), 2.76 (dd, J=13.3, 9.6 Hz, 1H), 1.68-1.54 (m, 3H), 0.94 (d, J=6.2Hz, 3H).

[0188] e. Acyl oxazolidinone IXe (R²=CH₂Ph; R⁸=CH₂Ph): By a procedureanalogous to that described for preparing acyl oxazolidinone IXb, thelithium anion of (S)-(−)-4-benzyl-2-oxazolidinone was treated withhydrocinnamoyl chloride to provide acyl oxazolidinone IXe in 82% yield.¹H NMR (300 MHz, CDCl₃) δ7.35-7.16 (m, 10H), 4.70-4.63 (m, 1H),4.21-4.14 (m, 2H), 3.38-3.19 (m, 3H), 3.08-2.98 (m, 2H), 2.75 (dd,J=13.4, 9.5 Hz, 1H).

EXAMPLE Acyl Oxazolidinones (X)

[0189] a. Acyl oxazolidinone Xb (R²=n-Pr; R⁸=CH₂Ph): A cold (0° C.)solution of acyl oxazolidinone IXb (5.74 g, 22.0 mmol) in 110 mLanhydrous CH₂Cl₂ was treated with 2.52 mL TiCl₄ (23.1 mmol) resulting inthe formation of an abundant precipitate. After 5 min,diisopropylethylamine (4.22 mL, 24.2 mmol) was added slowly and theresulting dark brown solution was stirred at room temperature for 35min. Benzyl chloromethyl ether (6.0 mL, 44.0 mmol) was rapidly added andthe mixture was stirred for 5 h at room temperature. 50 mL CH₂Cl₂ and 75mL of 10% aqueous NH₄Cl were then added, resulting in the formation ofyellow gummy material. After stirring the suspension vigorously for 10min, the supernatant was transferred in a separatory funnel and thegummy residue was taken up in 100 mL 1:1 10% aqueous NH₄Cl/CH₂Cl₂. Thecombined organic layers were then washed successively with 1N aqueousHCl, saturated NaHCO₃ and brine, dried over MgSO₄ and concentrated invacuo. The crude solid material was recrystallized from EtOAc/hexaneaffording 6.80 g of desired acyl oxazolidinone Xb as a white solid in81% yield. ¹H NMR (300MHz, CDCl₃), δ7.34-7.18(m, 10H), 4.77-4.69 (m,1H), 4.55 (s, 2H), 4.32-4.23 (m, 1H), 4.21-4.10 (m, 2H), 3.80 (t, J=9.0Hz, 1H), 3.65 (dd, J=9.0, 5.0 Hz, 1H), 3.23 (dd, J=13.5, 3.3 Hz, 1H),2.69 (dd, J=13.5, 9.3 Hz, 1H), 1.74-1.64 (m, 1H), 1.54-1.44 (m, 1H),1.40-1.28 (m, 2H), 0.91 (t, J=7.3 Hz, 3H). LRMS (FAB) m/e 382 (M+H⁺)

[0190] b. Acyl oxazolidinone Xa (R²=Et; R⁸=CH₂Ph): By a procedureanalogous to that described for preparing acyl oxazolidinone Xb, acyloxazolidinone Xa was obtained in 80% yield. ¹H NMR (300 MHz, CDCl₃)δ7.36-7.18 (m, 10H), 4.55 (s, 2H), 4.21-4.11 (m, 3H), 3.81 (t, J=9.0 Hz,1H), 3.66 (dd, J=9.0, 5.0 Hz, 1H), 3.23 (dd, J=13.5, 3.2 Hz, 1H), 2.70(dd, J=13.5, 9.3 Hz, 1H), 1.78-1.57 (m, 2H), 0.94 (t, J=7.5 Hz, 3H).

[0191] c. Acyl oxazolidinone Xc (R²=n-Bu; R⁸=CH₂Ph): By a procedureanalogous to that described for preparing acyl oxazolidinone Xb, acyloxazolidinone Xc was obtained in 91% yield. ¹H NMR (300 MHz, CDCl₃)δ7.38-7.17 (m, 10H), 4.72 (m, 1H), 4.54 (s, 2H), 4.27-4.10 (m, 2H), 3.79(t, J=8.7 Hz, 1H), 3.65 (dd, J=9.1, 5.0 Hz, 1H), 3.23 (dd, J=13.5, 3.3Hz, 1H), 2.68 (dd, J=13.5, 9.3 Hz, 1H), 1.75-1.68 (m, 1H), 1.31-1.26 (m,4H), 0.87 (t, J=6.8 Hz, 3H).

[0192] d. Acyl oxazolidinone Xd (R²=i-Bu; R⁸=CH₂Ph): By a procedureanalogous to that described for preparing acyl oxazolidinone Xb, acyloxazolidinone Xd was obtained in 98% yield. ¹H NMR (300 MHz, CDCl₃)δ7.38-7.17 (m, 10H), 4.75-4.67 (m, 1H), 4.57 (d, J=12.0 Hz, 1H), 4.51(d, J=12.0Hz, 1H), 4.41-4.36 (m, 1H), 4.20-4.09 (m, 2H), 3.74(t, J=9.0Hz, 1H), 3.65 (dd, J=9.0, 5.1 Hz, 1H), 3.23 (dd, J=13.5, 3.2 Hz, 1H),2.63 (dd, J=13.5, 9.5 Hz, 1H), 1.74-1.52 (m, 2H), 1.35 (dd, J=13.1, 6.1Hz, 1H), 0.92 (d, J=2.9 Hz, 3H), 0.90 (d, J=2.9 Hz, 3H).

[0193] e. Acyl oxazolidinone Xe (R²=CH₂Ph; R⁸=CH₂Ph): By a procedureanalogous to that described for preparing acyl oxazolidinone Xb, acyloxazolidinone Xe was obtained in 84% yield. ¹H NMR (300 MHz, CDCl₃)δ7.38-7.15 (m, 15H), 4.62-4.50 (m, 4H), 4.03 (dd, J=9.0, 2.7 Hz, 1H),3.93-3.82 (m, 2H), 3.66 (dd, J=9.2, 4.8 Hz, 1H), 3.19 (dd, J=13.5, 3.2Hz, 1H), 2.98 (dd, J=13.4, 8.2 Hz, 1H), 2.88 (dd, J=13.4, 7.3 Hz, 1H),2.68 (dd, J=13.5, 9.3 Hz, 1H).

EXAMPLE 3 Carboxylic Acids (XI)

[0194] a. Carboxylic acid Xb (R²=n-Pr): A cold (0° C.) solution of 6.60g (17.3 mmol) of acyl oxazolidinone Xb in 320 mL THF/H₂O was treatedsuccessively with 6.95 mL 35% aqueous H₂O₂ and a solution of lithiumhydroxide monohydrate (1.46 g, 34.6 mmol) in 20 mL H₂O. The mixture wasstirred for 16 h at 0° C. and then treated carefully first with asolution Na₂SO₃ (10.5 g) in 55 mL H₂O and then with a solution of NaHCO₃(4.35 g) in 100 mL H₂O. The mixture was stirred for 30 min at roomtemperature and concentrated in vacuo to remove the THF. The resultingaqueous mixture was then washed with CH₂Cl₂ (4×75 mL), cooled to 0° C.,acidified with 6N aqueous HCl and extracted with CH₂Cl₂ (1×200 mL and3×100 mL). The combined organic layers were then dried over MgSO₄ andconcentrated in vacuo affording 3.47 g (90%) of desired acid XIb as aclear colorless oil. ¹H NMR (300 MHz, CDCl₃) δ7.38-7.26 (m, 5H), 4.55(s, 2H), 3.67 (m, 1H), 3.57 (dd, J=9.2, 5.2 Hz, 1H), 2.75 (m, 1H),1.72-1.31 (m, 4H), 0.93 (t, J=7.2 Hz, 3H). LRMS (FAB) m/e 223 (M+H⁺)

[0195] b. Carboxylic acid XIa (R²=Et): By a procedure analogous to thatdescribed for preparing acyl oxazolidinone XIb, acyl oxazolidinone XIawas obtained in 48% yield. ¹H NMR (300 MHz, CDCl₃) δ7.36-7.27 (m, 5H),4.55 (s, 2H), 3.68 (dd, J=9.2, 7.9 Hz, 1H), 3.59 (dd, J=9.2, 5.4 Hz,1H), 2.68-2.65 (m, 1H), 1.71-1.62 (m, 2H), 0.97 (t, J=7.5 Hz, 3H).

[0196] c. Carboxylic acid XIc (R²=n-Bu): By a procedure analogous tothat described for preparing acyl oxazolidinone XIb, acyl oxazolidinoneXIc was obtained in 96% yield. ¹H NMR (300 MHz, CDCl₃) δ7.37-7.28 (m,5H), 4.55 (s, 2H), 3.67 (dd, J=9.1, 8.1 Hz, 1H), 3.57 (dd, J=9.2, 5.3Hz, 1H), 2.72 (m, 1H), 1.67-1.51 (m, 2H), 1.36-1.27 (m, 4H), 0.89 (t,J=6.9 Hz, 3H).

[0197] d. Carboxylic acid XId (R²=i-Bu): By a procedure analogous tothat described for preparing acyl oxazolidinone XIb, acyl oxazolidinoneXId was obtained in 80% yield. ¹H NMR (300 MHz, CDCl₃) δ7.37-7.28 (m,5H), 4.55 (s, 2H), 3.64 (t, J=9.1 Hz, 1H), 3.54 (dd, J=9.1, 5.1 Hz, 1H),2.81 (m, 1H), 1.68-1.54 (m, 2H), 1.36-1.27 (m, 1H), 0.92 (d, J=4.9 Hz,3H), 0.90 (d, J=4.9 Hz, 3H).

[0198] e. Carboxylic acid XIe (R²=CH₂Ph): By a procedure analogous tothat described for preparing acyl oxazolidinone XIb, acyl oxazolidinoneXIe was obtained in 92% yield. ¹H NMR (300 MHz, CDCl₃) δ7.38-7.16 (m,10H), 4.53 (d, J=12.1 Hz, 1H), 4.50 (d, J=12.1 Hz, 1H), 3.68-3.57 (m,2H), 3.09-2.85 (m, 3H).

EXAMPLE 4 Diethyl Amides (XII)

[0199] a. Diethylamide XIIb (R²=n-Pr; R⁵=R⁶=Et): A cooled solution (0°C.) of carboxylic acid XIb (3.40 g, 15.3 mmol) in 1:1 MeCN/CH₂Cl₂ (150mL), containing diethylamine (2.36 mL, 23.0 mmol) and2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU, 5.89 g, 18.4 mmol), was treated with diisopropylethylamine (6.7mL, 38.2 mmol) over 1.5 h (syringe pump). The mixture was thenconcentrated in vacuo and partitioned between ether (200 mL) and H₂O(100 mL). The aqueous layer was extracted with more ether (2×100 mL) andthe combined organic layers were washed with aqueous 1N HCl (3×50 mL),saturated aqueous NaHCO₃ and brine, dried over MgSO₄ and concentrated invacuo. Chromatographic purification (230-400 mesh SiO₂, elution with 1:3AcOEt/hexane) afforded 4.24 g (97%) of diethyl amide XIIb as a clearcolorless oil. ¹H NMR (300 MHz, CDCl₃) δ7.35-7.23 (m, 5H), 4.52 (d,J=12.0 Hz, 1H), 4.44 (d, J=12.0 Hz, 1H), 3.67 (t, J=8.6 Hz, 1H), 3.51(dd, J=8.7, 5.5 Hz, 1H), 3.46-3.27 (m, 4H), 2.96 (m, 1H), 1.67-1.57 (m,1H), 1.48-1.22 (m, 4H), 1.20-1.10 (m, 6H), 0.90 (t, J=7.2 Hz, 3H). LRMS(FAB) m/e 278 (M+H⁺)

[0200] b. Diethylamide XIIa (R²=Et; R⁵=R⁶=Et): By a procedure analogousto that described for preparing diethylamide XIIb, diethylamide XIIa wasobtained in 73% yield. ¹H NMR (300 MHz, CDCl₃) δ7.33-7.26 (m, 5H), 4.52(d, J=12.0 Hz, 1H), 4.44 (d, J=12.0 Hz, 1H), 3.68 (t, J=8.6 Hz, 1H),3.53-3.33 (m, 5H), 2.90(m, 1H), 1.75-1.50 (m, 2H), 1.18(t, J=7.1 Hz,3H), 1.13 (t, J=7.1 Hz, 3H), 0.89 (t, J=7.4 Hz, 3H).

[0201] c. Diethylamide XIIc (R²=n-Bu; R⁵=R⁶=Et): By a procedureanalogous to that described for preparing diethylamide XIIb,diethylamide XIIc was obtained in 94% yield. ¹H NMR (300 MHz, CDCl₃)δ7.35-7.25 (m, 5H), 4.51 (d, J=12.0 Hz, 1H), 4.44 (d, J=12.0 Hz, 1H),3.67 (t, J=8.6 Hz, 1H), 3.51 (dd, J=8.8, 5.5 Hz, 1H), 3.46-3.29 (m, 1H),2.94 (m, 1H), 1.66-1.62 (m, 2H), 1.33-1.10 (m, 9H), 0.85 (t, J=7.0 Hz,3H).

[0202] d. Diethylamide XIId (R²=i-Bu; R⁵=R⁶=Et): By a procedureanalogous to that described for preparing diethylamide XIIb,diethylamide XIId was obtained in 95% yield. ¹H NMR (300 MHz, CDCl₃)δ7.35-7.23 (m, 5H), 4.51 (d, J=12.0 Hz, 1H), 4.44 (d, J=12.0 Hz, 1H),3.65 (t, J=8.7 Hz, 1H), 3.54-3.28 (m, 5H), 3.03 (m, 1H), 1.63-1.49 (m,2H), 1.33-1.24 (m, 1H), 1.18 (t, J=7.1 Hz, 3H), 1.12 (t, J=7.1 Hz, 3H),0.90 (t, J=6.4 Hz, 3H).

[0203] e. Diethylamide XIIe (R²=CH₂Ph; R⁵=R⁶=Et): By a procedureanalogous to that described for preparing diethylamide XIIb,diethylamide XIIe was obtained in 89% yield. ¹H NMR (300 MHz, CDCl₃)δ7.35-7.16 (m, 10H), 4.53 (d, J=12.1 Hz, 1H), 4.47 (d, J=12.1 Hz, 1H),3.77 (t, J=8.5 Hz, 1H), 3.59 (dd, J=8.8, 5.7 Hz, 1H), 3.40 (m, 1H),3.22-2.89 (m, 5H), 2.79 (dd, J=13.0, 5.1 Hz, 3H), 1.01 (t, J=7.1 Hz,3H), 0.85 (t, J=7.2 Hz, 3H).

EXAMPLE 5 Alcohols (XIII)

[0204] a. Alcohol XIIIb (R²=n-Pr; R⁵=R⁶=Et): To a solution ofdiethylamide XIIb (4.08 g, 14.7 mmol) in 140 mL MeOH was added 20%Pd(OH)₂/C (400 mg) and the suspension was hydrogenated at atmosphericpressure and room temperature for 15 h. Filtration of the catalyst andconcentrating the filtrate in vacuo afforded 2.84 g (100%) of thedesired primary alcohol XIIIb. ¹H NMR (300 MHz, CDCl₃) δ3.74 (br. d,J=4.2 Hz, 1H), 3.61-3.15 (m, 5H), 2.71 (m, 1H), 1.69-1.24 (m, 4H), 1.20(t, J=7.1 Hz, 3H), 1.12 (t, J=7.1 Hz, 3H), 0.92 (t, J=7.2 Hz, 3H). LRMS(FAB) m/e 188 (M+H⁺).

[0205] b. Alcohol XIIIa (R²=Et; R⁵=R⁶=Et): By a procedure analogous tothat described for preparing alcohol XIIb, alcohol XIIIa was obtained in100% yield. ¹H NMR (300 MHz, CDCl₃) δ3.76 (m, 2H), 3.58-3.19 (m, 4H),2.64 (m, 1H), 1.71-1.65 (m, 2H), 1.21 (t, J=7.1 Hz, 3H), 1.13 (t, J=7.1Hz, 3H), 0.96 (t, J=7.4 Hz, 3H).

[0206] c. Alcohol XIIIc (R²=n-Bu; R⁵=R⁶=Et): By a procedure analogous tothat described for preparing alcohol XIIIb, alcohol XIIIc was obtainedin 100% yield. ¹H NMR (300 MHz, CDCl₃) δ3.76 (d, J=4.5 Hz, 2H),3.58-3.19 (m, 4H), 2.72-2.65 (m, 2H), 1.68-1.55 (m, 2H), 1.40-1.24 (m,4H), 1.20 (t, J=7.1 Hz, 3H), 1.12 (t, J=7.1 Hz, 3H), 0.90 (t, J=6.9Hz,3H).

[0207] d. Alcohol XIIId (R²=i-Bu; R⁵=R⁶=Et): By a procedure analogous tothat described for preparing alcohol XIIIb, alcohol XIIId was obtainedin 100% yield. ¹H NMR (300 MHz, CDCl₃) δ3.78-3.68 (m, 2H), 3.57-3.15 (m,4H), 2.81-2.73 (m, 1H), 1.70-1.60(m, 2H), 1.40-1.28(m, 1H), 1.21 (t,J=7.1 Hz, 3H), 1.12 (t, J=7.1 Hz, 3H), 0.92 (m, 6H).

[0208] e. Alcohol XIIIe (R²=CH₂Ph; R⁵=R⁶=Et): By a procedure analogousto that described for preparing alcohol XIIIb, alcohol XIIIe wasobtained in 100% yield. ¹H NMR (300 MHz, CDCl₃) δ7.29-7.16 (m, 5H),3.81-3.71 (m, 2H), 3.61-3.50 (m, 1H), 3.15-2.87 (m, 6H), 1.05 (t, J=7.1Hz, 3H), 0.98 (t, J=7.1 Hz, 3H).

EXAMPLE 6 Aldehydes (XIV)

[0209] a. Aldehyde XIVb (R²=n-Pr; R⁵=R⁶=Et): To a solution of alcoholXIIIb (2.34 g, 12.7 mmol) in wet CH₂Cl₂ (125 mL, prepared by stirringCH₂Cl₂ with water and separating the organic layer) was addedDess-Martin periodinane (8.06 g, 19.0 mmol). The mixture was stirred atroom temperature for 40 min and was then poured into a mixture of 5%aqueous Na₂S₂O₃ (250 mL) containing 5.2 g NaHCO₃, and ether (200 mL).The biphasic mixture was stirred vigorously for 5 min and the aqueouslayer was extracted with 15% CH₂Cl₂/Et₂O (2×100 mL). The combinedorganic layers were then washed with H₂O (3×75 mL) and brine, dried overMgSO₄, filtered and concentrated in vacuo to afford 2.06 g (88%) ofdesired aldehyde XIVb, a clear colorless oil. ¹H NMR (300 MHz, CDCl₃)δ9.60 (d, J=3.5 Hz, 1H), 3.49-3.30 (m, 5H), 1.96-1.85 (m, 2H), 1.39-1.31(m, 2H), 1.19 (t, J=7.1 Hz, 3H), 1.13 (t, J=7.1 Hz, 3H), 0.95 (t, J=7.3Hz, 3H).

[0210] b. Aldehyde XIVb (R²=n-Pr; R⁵=R⁶=Et): To a solution of crudeXIIIb (1.25 g, 6.68 mmol) in a mixture of toluene (20 mL), ethyl acetate(20 mL), and water (3 mL) was added 2,2,6,6-tetramethyl-1-piperidinyloxy(TEMPO), free radical (9 mg). The mixture was cooled to 0° C. and asodium hypochlorite solution, prepared by adding 4.3 mL of aqueoussodium hypochlorite (10-13% available chlorine) to 1.6 g of NaHCO₃ in 20mL of water, was added by portions over a period of 30 min. Sodiumbromide (660 mg) was added and the solution turned pale orange. Within afew minutes the color of the reaction mixture returned to off-white.Additional sodium hypochlorite (4.7 mL) was added in several portions todrive the reaction to completion. The aqueous layer was separated andextracted with toluene (20 mL) and ethyl acetate (2×20 mL). The combinedorganic extract was washed with a solution of KI (70 mg) in 10% aqueousKHSO₄. The organic layer was then washed with 5% Na₂S₂O₃ and pH 7phosphate buffer, dried (Na₂SO₄), and concentrated to give XIVb as apale yellow oil (1.1 g). Spectral data for this compound matched thatfor the product from Example 6a above.

[0211] c. Aldehyde XIVa (R²=Et; R⁵=Rf =Et): By a procedure analogous tothat described for preparing alcohol XIVb, aldehyde XIVa was obtained in80% yield. ¹H NMR (300 MHz, CDCl₃) δ9.61 (d, J=3.6 Hz, 1H), 3.48-3.29(m, 5H), 2.02-1.90 (m, 2H), 1.19 (t, J=7.1 Hz, 3H), 1.14 (t, J=7.1 Hz,3H), 0.96 (t, J=7.4 Hz, 3H).

[0212] d. Aldehyde XIVc (R²=n-Bu; R⁵=R⁶=Et): By a procedure analogous tothat described for preparing alcohol XIVb, aldehyde XIVc was obtained in98% yield. H NMR (300 MHz, CDCl₃) δ9.59 (d, J=3.6 Hz, 1H), 3.48-3.29 (m,5H), 1.97-1.87 (m, 2H), 1.39-1.22 (m, 4H), 1.18 (t, J=7.2 Hz, 3H), 1.13(t, J=7.2 Hz, 3H), 0.90 (t, J=7.0 Hz, 3H).

[0213] e. Aldehyde XIVd (R²=i-Bu; R¹=R⁶=Et): By a procedure analogous tothat described for preparing alcohol XIVb, aldehyde XIVd was obtained in96% yield. ¹H NMR (300 MHz, CDCl₃) δ9.57 (d, J=3.7 Hz, 1H), 3.51-3.27(m, 5H), 1.83 (t, J=7.1 Hz, 3H), 1.66-1.55 (m, 1H), 1.20 (t, J=7.1 Hz,3H), 1.13 (t, J=7.1 Hz, 3H), 0.93 (d, J=6.6 Hz, 6H).

[0214] f. Aldehyde XIVe (R²=CH₂Ph; R¹=R⁶=Et): By a procedure analogousto that described for preparing alcohol XIVb, aldehyde XIVe was obtainedin 97% yield. ¹H NMR (300 MHz, CDCl₃) δ9.69 (d, J=2.9 Hz, 1H), 7.29-7.16(m, 5H), 3.65 (m, 1H), 3.53-3.42 (m, 1H), 3.30 (dd, J=13.5, 9.3 Hz, 1H),3.23-3.13 (m, 2H), 3.06-2.91 (m, 2H), 1.04 (t, J=7.1 Hz, 3H), 0.93 (t,J=7.1 Hz, 3H).

[0215] The preparation of clasto-lactacystin β-lactone and analogsthereof according to the synthetic scheme outlined in Scheme 1 asexemplified in Examples 7-9.

EXAMPLE 7 Aldol adducts (II)

[0216] a. Aldol adduct IIb (R²=n-Pr; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et):To a cold (−78° C.) solution of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) inether (35 mL) was added lithium bis(trimethylsilyl)amide (2.17 of a 1 Msolution in hexane, 2.17 mmol). After 30 min, the orange solution wastreated dropwise with a 1M solution of dimethylaluminum chloride inhexane (4.55 mL, 4.55 mmol) and the mixture was stirred for another 60min before being cooled down to −85° C. (liquid N₂ was added to the dryice/acetone bath). A solution of aldehyde XIVb (420 mg, 2.27 mmol) inether (4 mL) was then added over 10 min along the side of the flask. Themixture was then allowed to warm up to −40° C. over 2.5 h and thenquenched by adding 35 mL of saturated aqueous NH₄Cl and 25 mL AcOEt.Enough 2 N HCl was then added until 2 clear phases were obtained (ca. 15mL added). The aqueous layer was extracted with AcOEt (2×20 mL) and thecombined organic layers were washed successively with 0.5 N aqueous HCl(20 mL), H₂O (20 mL), 0.5 M aqueous NaHSO₃ (2×15 mL), saturated aqueousNaHCO₃ and finally with brine, then dried over Na₂SO₄ and concentratedin vacuo affording 879 mg (>100%) of crude Aldol product IIb which waspure enough to be used directly in the subsequent step. ¹H NMR (300 MHz,CDCl₃) δ8.02-7.97 and 7.53-7.39 (m, 5H), 6.58 (d, J=9.9 Hz, 1H), 4.82(d, J=2.4 Hz, 1H), 3.73 (s, 3H), 3.69-3.61 (m, 2H), 3.49-3.39 (m, 2H),3.24-3.16 (m, 1H), 3.05 (m, 1H), 2.89 (m, 1H), 2.28-2.23 (m, 1H),1.98-1.91 (m, 1H), 1.37-1.20 (m, 6H), 1.19-1.06 (m, 6H), 0.87 (t, J=7.1Hz, 3H), 0.70 (d, J=6.7Hz, 3H).

[0217] Aldol product IIb was also obtained in 100% yield by a procedureanalogous to that described above but using cis-oxazoline Ib (see below)instead of trans-oxazoline Ia.

[0218] b. Aldol adduct IIb (R²=n-Pr; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et):To a cold (−78° C.) solution of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph)(20.74 g) in THF (280 mL) was added lithium bis(trimethylsilyl)amide(92.4 mL of a 1 M solution in hexane) over 75 min. After 30 min, theorange solution was treated dropwise with a 1M solution ofdimethylaluminum chloride in hexane (202 mL) and the mixture was stirredfor another 40 min before being cooled down to −85° C. (liquid N₂ wasadded to the dry ice/acetone bath). A solution of aldehyde XIVb (19.43g) in THF (50 mL) was then added over 45 min. The mixture was thenallowed to warm to −50° C. over 40 min and then to −20° C. over 25 min.The yellow reaction mixture was again cooled to −78° C. and thenquenched by cautious addition of40 mL of saturated aqueous NH₄Cl. Thereaction mixture was poured slowly into 460 mL of saturated aqueousNH₄Cl. AcOEt (500 mL) was added, and with good stirring the reactionmixture was acidifed with 6 N HCl to produce two clear phases. Theaqueous layer was extracted with AcOEt (2×200 mL), and the combinedorganic layers were washed successively with H₂O (2×200 mL), saturatedaqueous NaHCO₃ (2×200 mL), and brine (2×300 mL). The organic extract wasdried over Na₂SO₄ and MgSO₄ and concentrated in vacuo to afford 41.55 gof crude Aldol product IIb which was pure enough to be used directly inthe subsequent step. Spectral data for this compound matched that forthe product from Example 7a above.

[0219] c. Aldol adduct Ia (R²=Et; R=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et): By aprocedure analogous to that described for preparing Aldol adduct IIb,the lithium anion of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) was treatedsuccessively with dimethylaluminum chloride and aldehyde XIVa to provideAldol adduct IIa in 95% yield. ¹H NMR (300 MHz, CDCl₃) δ8.00-7.97 and7.51-7.39 (m, 5H), 6.50 (d, J=9.9 Hz, 1H), 4.80 (d, J=2.4 Hz, 1H),3.81-3.64 (m, 2H), 3.74 (s, 3H), 3.45 (m, 2H), 3.19 (m, 2H), 2.93-2.84(m, 2H), 2.24 (m, 1H), 1.89 (m, 1H), 1.73-1.64 (m, 4H), 1.29 (t, J=7.2Hz, 3H), 1.12 (d, J=6.9 Hz, 3H), 1.07 (d, J=7.2 Hz, 3H), 0.70 (d, J=6.7Hz, 3H).

[0220] d. Aldol adduct IIc (R²=n-Bu; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et).By a procedure analogous to that described for preparing Aldol adductIIb, the lithium anion of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) wastreated successively with dimethylaluminum chloride and aldehyde XIVc toprovide Aldol adduct IIc in 100% yield. ¹H NMR (300 MHz, CDCl₃)δ8.02-7.98 and 7.53-7.33 (m, 5H), 6.57 (d, J=10.0 Hz, 1H), 4.81 (d,J=2.3 Hz, 1H), 3.73 (s, 3H), 3.68-3.60 (m, 2H), 3.49-3.17 (m, 2H), 3.00(m, 1H), 2.90 (m, 1H), 1.98-1.87 (m, 2H), 1.38-0.83 (m, 16H), 0.70 (d,J=6.7 Hz, 3H).

[0221] e. Aldol adduct IId (2=i-Bu; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et): Bya procedure analogous to that described for preparing Aldol adduct IIb,the lithium anion of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) was treatedsuccessively with dimethylaluminum chloride and aldehyde XIVd to provideAldol adduct IId in 100% yield. ¹H NMR (300 MHz, CDCl₃) δ8.01-7.80 and7.55-7.20 (m, 5H), 4.87 (d, J=2.3 Hz, 1H), 3.73 (s, 3H), 3.69-3.58 (m,2H), 3.51-3.32 (m, 2H), 2.98-2.87 (m, 1H), 2.33-2.24 (m, 1H), 2.12-2.02(m, 1H), 1.83 (t, J=7.1 Hz, 1H), 1.35 (t, J=7.1 Hz, 3H), 1.25-1.05 (m,5H), 0.93 (d, J=6.6 Hz, 3H), 0.89 (d, J=6.5 Hz, 3H), 0.80 (d, J=6.5 Hz,3H), 0.69 (d, J=6.7 Hz, 3H).

[0222] f. Aldol adduct IIe (R²=CH₂Ph; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et):By a procedure analogous to that described for preparing Aldol adductIIb, the lithium anion of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) wastreated successively with dimethylaluminum chloride and aldehyde XIVe toprovide Aldol adduct IIe in 100% yield. ¹H NMR (300 MHz, CDCl₃)δ8.01-7.93 and 7.54-7.10 (m, 10H), 4.71 (d, J=2.5 Hz, 1H), 3.73 (s, 3H),3.68-3.58 (m, 2H), 3.48-2.79 (m, 6H), 2.17 (m, 1H), 1.12-0.91 (m, 9H),0.68 (d, J=6.7 Hz, 3H).

EXAMPLE 8 γ-Lactams (IV)

[0223] a. γ-Lactam IVb (R²=n-Pr; R¹=i-Pr; R³=Me): A solution of Aldoladduct IIb (4.72 g, 10.9 mmol) in 100mL 1:9 AcOH/MeOH, to which wasadded 4.8 g 20% Pd(OH)₂/C, was vigorously shaken under 55 p.s.i. H₂ for60 h. The mixture was brought down to atmospheric temperature beforebeing filtered and concentrated in vacuo. The solid obtained waspurified by flash chromatography (SiO₂, elution with 1% AcOH in 1:1AcOEt/hexane) affording 2.23 g (75%) of desired γ-lactam IVb as a whitesolid. ¹H NMR (300 MHz, CDCl₃) δ7.89 (br. s, 1H), 4.77 (br. d, J=11.5Hz, 1H), 4.47 (dd, J=11.5, 5.6 Hz, 1H), 4.08 (dd, J=9.4, 5.0 Hz, 1H),3.83 (s, 3H), 2.93 (m, 1H), 1.78-1.39 (m, 6H), 1.02-0.88 (m, 9H).

[0224] b. γ-Lactam IVa (R²=Et; R¹=i-Pr; R³=Me): By a procedure analogousto that described for preparing γ-lactam IVb, Aldol adduct IIa washydrogenated at 55 p.s.i. for 48 h to provide γ-lactam IVa in 72% yield.¹H NMR (300 MHz, CDCl₃) δ7.79 (br. s, 1H), 4.62 (br. d, J=11.2 Hz, 1H),4.51 (dd, J=11.2, 5.4 Hz, 1H), 3.83 (s, 3H), 2.85 (m, 11H), 1.77-1.64(m, 3H), 1.01 (t, J=7.4 Hz, 3H), 0.98 (d, J=6.9 Hz, 3H), 0.95 (d, J=6.9Hz, 3H).

[0225] c. γ-Lactam IVc (R²=n-Bu; R¹=i-Pr; R³=Me): A solution of Aldoladduct IIc (361 mg, 0.80 mmol) in 6 mL 1:9 AcOH/MeOH, to which was added250 mg 20% Pd(OH)₂/C, was vigorously shaken under 50 p.s.i. H₂ for 24 h.More catalyst (100 mg) was then added and the mixture was again shakenat 50 p.s.i. for another 24 h after which time it brought down toatmospheric temperature before being filtered. The filtrate was thenheated to reflux for 30 min, cooled to room temperature and concentratedin vacuo. The solid obtained was co-evaporated once with toluene andpurified by flash chromatography (SiO₂, elution with 4% MeOH/CHCl₃)affording 140 mg (61%) of desired γ-lactam IVc as a white solid. ¹H NMR(300 MHz, CDCl₃) δ8.02 (br. s, 1H), 4.93 (br. d, J=11.3 Hz, 1H), 4.46(dd, J=11.3, 5.5 Hz, 1H), 4.15-4.08 (m, 1H), 3.83 (s, 3H), 2.94-2.87 (m,1H), 1.80-1.34 (m, 6H), 0.94 (d, J=6.9 Hz, 3H), 0.89 (t, J=7.2 Hz, 3H).

[0226] d. γ-Lactam IVd (R²=i-Bu; R¹=i-Pr; R³=Me): By a procedureanalogous to that described for preparing γ-lactam IVe, Aldol adduct IIdwas hydrogenated at 50 p.s.i. for 40 h and heated to reflux for 30 minproviding γ-lactam IVd in 61% yield. ¹H NMR (300 MHz, CDCl₃) δ7.92 (br.s, 1 H), 4.81 (br. d, J=11.5 Hz, 1H), 4.46 (m, 1H), 4.09 (m, 1H), 3.83(s, 3H), 3.04-2.98 (m, 1H), 1.78-1.73 (m, 2H), 1.66-1.47 (m, 3H),1.00-0.90 (m, 12H).

[0227] e. γ-Lactam IVe (R²=CH₂Ph; R¹=i-Pr; R³=Me): By a procedureanalogous to that described for preparing γ-lactam IVc, Aldol adduct IIewas hydrogenated at 50 p.s.i. for 24 h and heated to reflux for 30 minproviding γ-lactam IVe in 71% yield. ¹H NMR (300 MHz, CDCl₃) δ8.01 (br.s, 1H), 7.35-7.15 (m, 5H), 5.02 (br. d, J=11.7 Hz, 1H), 4.40-4.34 (m,1H), 4.06-4.01 (m, 1H), 3.84 (s, 3H), 3.34-3.27 (m, 1H), 3.10-3.04 (m,2H), 1.84-1.72 (m, 1H), 0.98 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.9 Hz, 3H).

EXAMPLE 9 β-Lactones (VII)

[0228] a. β-Lactone VIIb (R²=n-Pr; R¹=i-Pr): To a cold (0° C.) solutionof γ-lactam IVb (2.20 g, 8.06 mmol) in EtOH (100 mL) was added 0.1Naqueous NaOH (100 mL, 10.0 mmol). The mixture was stirred at roomtemperature for 15 h after which time H₂O (50 mL) and AcOEt (100 mL)were added. The aqueous layer was then washed with AcOEt (2×50 mL),acidified with 6N aqueous HCl and concentrated in vacuo to a volume ofca 60 mL. This solution was then frozen and lyophilized. The obtainedsolid was suspended in THF, filtered to get rid of sodium chloride andconcentrated in vacuo affording 2.05 g (98%) of the desireddihydroxyacid as white solid. ¹H NMR (300 MHz, CD₃OD) δ4.42 (d, J=5.8Hz, 1H), 3.90 (d, J=6.5 Hz, 1H), 2.84 (m, 1H), 1.70-1.24 (m, 6H),0.95-0.84 (m, 9H).

[0229] To a solution of the dihydroxyacid (1.90 g, 7.33 mmol) inanhydrous THF (36 mL) was added a solution of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU, 2.59, 8.06 mmol) in anhydrous MeCN (36 mL) followed bytriethylamine (0.72 mL, 22.0 mmol). After stirring for 70 min at roomtemperature, some toluene was added and the mixture was concentrated invacuo and co-evaporated 2 more times with toluene. Purification by flashchromatography (SiO₂, elution with 2:3 AcOEt/hexane) afforded 1.44 g(81%) of desired β-lactone VIb as a white solid. ¹H NMR (300 MHz, CDCl₃)δ6.07 (br. s, 1H), 5.26 (d, J=6.1 Hz, 1H), 3.97 (dd, J=6.4, 4.4 Hz, 1H),2.70-2.63 (m, 1H), 2.03 (d, J=6.4 Hz, 3H), 1.93-1.44 (m, 5H), 1.07 (d,J=7.0 Hz, 3H), 0.99 (d, J=7.3 Hz, 3H), 0.91 (d, J=6.7 Hz, 3H). LRMS(FAB) m/e 242 (M+H⁺).

[0230] b. β-Lactone VIIa (R²=Et; R¹=i-Pr): Hydrolysis of IVa, asdescribed for IVb above, afforded the corresponding dihydroxyacid in100% yield. ¹H NMR (300 MHz, CD₃OD) δ4.45 (d, J=5.8 Hz, 1H), 3.90 (d,J=6.4 Hz, 1H), 2.74 (m, 1H), 1.71-1.53 (m, 3H), 0.94 (t, J=7.4 Hz, 3H),0.92 (d, J=6.8 Hz, 3H), 0.88 (d, J=6.8 Hz, 3H).

[0231] By a procedure analogous to that described for preparingβ-lactone VIIb, β-lactone VIIa was obtained in 79% yield. ¹H NMR (300MHz, CDCl₃) δ6.17 (br. s, 1H), 5.30 (d, J=6.0 Hz, 1H), 3.98 (dd, J=6.4,4.4 Hz, 1H), 2.60 (m, 1H), 2.08 (d, J=6.4 Hz, 3H), 1.97 (m, 2H), 1.75(m, 1H), 1.12 (t, J=7.5 Hz, 3H), 1.07 (d, J=6.8 Hz, 3H), 0.92 (d, J=6.8Hz, 3H).

[0232] c. β-Lactone VIIc (R²=n-Bu; R¹=i-Pr): Hydrolysis of IVe, asdescribed for IVb above, afforded the corresponding dihydroxyacid in100% yield. ¹H NMR (300 MHz, CD₃OD) δ4.42 (d, J=5.8 Hz, 1H), 3.90 (d,J=6.4 Hz, 1H), 2.86-2.79 (m, 1H), 1.70-1.24 (m, 8H), 0.97-0.86 (m, 9H).

[0233] By a procedure analogous to that described for preparingβ-lactone VIIb, β-lactone VIIc was obtained in 40% yield. ¹H NMR (300MHz, CDCl₃) δ6.14 (br. s, 1H), 5.27 (d, J=6.1 Hz, 1H), 3.97 (d, J=4.4Hz, 1H), 2.68-2.61 (m, 1H), 1.94-1.86 (m, 2H), 1.72-1.36 (m, 7H), 1.07(d, J=7.0 Hz, 3H), 0.93 (t, J=7.1 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H). LRMS(FAB) m/e 256 (M+H⁺)

[0234] d. β-Lactone VIId (R²=i-Bu; R¹=i-Pr): Hydrolysis of IVd, asdescribed for IVb above, afforded the corresponding dihydroxyacid in100% yield. ¹H NMR (300 MHz, CD₃OD) δ4.50 (d, J=5.8 Hz, 1H), 4.00 (d,J=6.5 Hz, 1H), 3.09-3.02 (m, 1H), 1.90-1.61 (m, 3H), 1.49-1.40 (m, 2H),1.02 (d, J=6.7 Hz, 3H), 0.98 (d, J=6.5 Hz, 3H), 0.97 (d, J=6.7 Hz, 3H).

[0235] By a procedure analogous to that described for preparingβ-lactone VIIb, β-lactone VIId was obtained in 62% yield. ¹H NMR (300MHz, CDCl₃) δ6.16 (br. s, 1H), 5.25 (d, J=6.1 Hz, 1H), 3.97 (d, J=4.4Hz, 1H), 2.71 (dd, J=15.1, 6.2 Hz, 1H), 1.95-1.66 (m, 5H), 1.08 (d,J=6.9 Hz, 3H), 0.99 (d, J=6.3 Hz, 3H), 0.98 (d, J=6.3 Hz, 3H), 0.92 (d,J=6.7 Hz, 3H). LRMS (FAB) m/e 256 (M+H⁺).

[0236] e. β-Lactone VIIe (R²=CH₂Ph; R¹=i-Pr): Hydrolysis of IVe, asdescribed for IVb above, afforded the corresponding dihydroxyacid in 88%yield. ¹H NMR (300 MHz, CD₃OD) δ7.25-7.04 (m, 5H), 4.29 (d, J=5.7 Hz,1H), 3.83 (d, J=6.4 Hz, 1H), 3.01-2.82 (m, 3H), 1.65 (m, 1H), 0.90 (d,J=6.6 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H).

[0237] By a procedure analogous to that described for preparingβ-lactone VIIb, β-lactone VIIe was obtained in 77% yield. ¹H NMR (300MHz, CDCl₃) δ7.36-7.20 (m, 5H), 6.57 (br. s, 1H), 5.08 (d, J=5.4 Hz,1H), 3.94 (d, J=4.5 Hz, 1H), 3.25 (d, J=10.1 Hz, 1H), 3.01-2.89 (m, 2H),1.92-1.81 (m, 1H), 1.05 (d, J=6.9 Hz, 3H), 0.86 (d, J=6.7 Hz, 3H). LRMS(FAB) m/e 290 (M+H⁺).

[0238] The preparation of cis-oxazolines and trans-oxazolines accordingto the synthetic schemes illustrated in Schemes 3 and 4 as illustratedby Examples 10 and 11.

EXAMPLE 10 cis-Oxazoline (Ia)

[0239] a. Ethyl 3-(isopropyl)propenoate (XV; R¹=i-Pr; R³=Me): To astirred solution of carbomethoxymethylene triphenylphosphorane (56.04 g,167.6 mmol) in dry CH₂Cl₂ (168 mL) at 0° C. was added dropwiseisobutyraldehyde (17.4 mL, 191.6 mmol). After 5 min, the reactionmixture was warmed to room temperature and stirred for 24 h. The solventwas removed in vacuo and pentane was added to the white oily solid toprecipitate triphenylphosphine oxide. The solid was filtered off and thefiltrate concentrated in vacuo. The procedure was repeated one more timeand the crude olefin (20.00 g, 93%) was obtained as a yellow oil thatwas sufficiently pure for the next step. ¹H NMR (300 MHz, CDCl₃) δ6.95(dd, J=15.7, 6.6 Hz, 1H), 5.77 (dd, J=15.7, 1.5 Hz), 3.72 (s, 3H), 2.44(m, 1H), 1.06 (d, J=6.7 Hz, 6H).

[0240] b. (2S,3R)-Methyl 2,3-dihydroxy-3-[isopropyl]propionate (XVIa;R¹=i-Pr; R³=Me): A mixture of AD-mix-β (100.00 g,), methanesulfonamide(6.78 g, 71.3 mmol) and tert-butanol-water (1:1, 720 mL) was stirredvigorously at room temperature for 5 min. The reaction mixture was thencooled to 0° C. and α,β-unsaturated ester XV (R¹=i-Pr; R³=Me) (9.14 g,71.3 mmol) was added dropwise via a Pasteur pipette. After stirring at0° C. for 96 h, Na₂SO₃ (3.0 g) was added, and stirring continued at roomtemperature for 1 h. The mixture was diluted with ethyl acetate (200 mL)and transferred to a separatory funnel. The organic layer was removedand the aqueous phase extracted with ethyl acetate (2×100 mL). Thecombined organic layers were dried (Na₂SO₄), filtered, and concentratedin vacuo. The yellow oil obtained was passed through a silica gel padusing 1:1 hexane/ethyl acetate affording diol XVIa (R¹=i-Pr; R³=Me)(11.48 g, 94%) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ4.28 (dd,J=5.6, 1.8 Hz, 1H), 3.80 (s, 3H), 3.48 (m, 1H), 3.28 (m, 1H), 2.33 (d,J=9.3 Hz, 1H), 1.87 (m, 1H), 1.02 (d, J=6.7 Hz, 3H), 0.95 (d, J=6.7 Hz,3H).

[0241] c. (2R,3R)-Methyl 2-bromo-3-dihydroxy-3-(isopropyl)propionate(XVIIa; R¹=i-Pr; R³=Me): (2S,3R)-Methyl2,3-dihydroxy-3-[isopropyl]propionate XVIa (R¹=i-Pr; R³=Me) (1.0 g, 6.17mmol) and trimethylorthobenzoate (1.02 mL, 80.1 mmol) were dissolved inCH₂Cl₂ (20 mL) and treated with BF₃-OEt₂ (40.0 μL, 0.32 mmol). Afterstirring for 75 min, the mixture was concentrated under full vacuum(0.05 mm Hg) for 35 min. The mixture was redissolved in CH₂Cl₂ (20.0mL), cooled to 0° C. and treated sequentially with Et₃N (43.0 μL, 0.31mmol) and acetyl bromide (0.48 mL, 6.49 mmol). After stirring for 4 h at0° C., the reaction mixture was treated with saturated NaHCO₃ solution(12 mL) and allowed to warm up to room temperature. The layers wereseparated and the aqueous layer was extracted with CH₂Cl₂ (2×20 mL). Thecombined organic layers were dried (Na₂SO₄), filtered and concentratedin vacuo affording the crude α-bromo β-benzoate XVIIa (R¹=i-Pr; R³=Me)(1.36 g, 85%) as a clear colorless oil. ¹H NMR (300 MHz, CDCl₃)δ8.05-8.00 (m, 2H), 7.47-7.40 (m, 3H), 5.57 (dd, J=8.8, 3.9 Hz, 1H),4.47 (d, J=8.8 Hz, 1H), 3.67 (s, 3H), 2.45 (m, 1H), 1.01 (d, J=6.8 Hz,6H).

[0242] d. (2S3R)-Methyl 2-azo-3-dihydroxy-3-[isopropyl]propionate(XVIIIa; R¹=i-Pr; R³=Me): A solution of (2R,3R)-Methyl2-bromo-3-dihydroxy-3-[isopropyl]propionate XVIIa (R¹=i-Pr; R³=Me) (2.00g, 6.07 mmol) in 15 mL DMSO was treated with sodium azide (790.0 mg,12.2 mmol). After stirring for 12 h at room temperature, the mixture waspartitioned between H₂O and ethyl acetate (50 mL each). The aqueouslayer was extracted with more ethyl acetate and the combined organiclayers were dried over MgSO₄ and concentrated in vacuo affording thedesired α-azo β-benzoate (1.55 g, 87%) as a yellow oil. ¹H NMR (300 MHz,CDCl₃) δ8.07-8.02 (m, 2H), 7.55-7.43 (m, 3H), 5.40 (dd, J=8.8, 2.8 Hz,1H), 3.73 (s, 3H), 2.24 (m, 1H), 1.04 (d, J=5.8 Hz, 3H), 0.98 (d, J=5.8Hz, 3H).

[0243] Repeating the same procedure but using DMF as the solvent insteadof DMSO afforded the desired α-azo β-benzoate in 85% yield.

[0244] e. Benzamide XIXa (R¹=i-Pr; R³=Me): A solution of (2S,3R)-Methyl2-azo-3-dihydroxy-3-[isopropyl]propionate XVIIIa (R¹=i-Pr; R³=Me) (1.50g, 5.15 mmol) in ethyl acetate (25 mL) was treated with 200 mg of 20%Pd(OH)₂/C and the suspension was stirred vigorously in a H₂ atmosphereunder balloon pressure. After 12 hours, the mixture was filtered andrefluxed for 4 hours to complete the migration of the benzoyl group. Themixture was then cooled to room temperature and concentrated in vacuoaffording the desired benzamide (1.25 g, 92%) as a yellow oil. ¹H NMR(300 MHz, CDCl₃) δ7.85-7.83 (m, 2H), 7.46-7.40 (m, 3H), 6.99 (br. d,J=9.1 Hz, 1H), 5.05 (dd, J=9.1, 1.9 Hz, 1H), 3.77 (s, 3H), 1.79 (m, 1H),1.03 (d, J=6.7 Hz, 3H), 0.99 (d, J=6.7 Hz, 3H).

[0245] f. cis-Oxazoline Ia (R¹=i-Pr; R³=Me): A solution of 500 mg ofbenzamide XIXa (R¹=i-Pr; R³=Me) (18.8 mmol) in CH₂Cl₂ (20 mL) wastreated with 4.50 mL thionyl chloride (61.7 mmol). After stirring atroom temperature for 24 h, the mixture was diluted with CH₂Cl₂ andwashed with saturated NaHCO₃ solution, dried (Na₂SO₄), concentrated invacuo and chromatographed (silica gel, 1:1 hexane/ethyl acetate)affording the desired cis-oxazoline (248 mg, 53%) as a pale yellow oil.¹H NMR (300 MHz, CDCl₃) δ8.01-7.97 (m, 2H), 7.52-7.38 (m, 3H), 4.94 (d,J=9.8 Hz, 1H), 4.53 (dd, J=9.8, 7.8 Hz, 1H), 3.76 (s, 3H), 2.09 (m, 1H),1.05 (d, J=6.5 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H).

EXAMPLE 11 trans-Oxazoline (Ib)

[0246] a. Ethyl 3-(isopropyl)propenoate (XV; R¹=i-Pr; R³=Me): To astirred solution of carbomethoxymethylene triphenylphosphorane (56.04 g,167.6 mmol) in dry CH₂Cl₂ (168 mL) at 0° C. was added dropwiseisobutyraldehyde (17.4 mL, 191.6 mmol). After 5 min, the reactionmixture was warmed to room temperature and stirred for 24 h. The solventwas removed in vacuo and pentane was added to the white oily solid toprecipitate triphenylphosphine oxide. The solid was filtered off and thefiltrate concentrated in vacuo. The procedure was repeated one more timeand the crude olefin (20.00 g, 93%) was obtained as a yellow oil thatwas sufficiently pure for the next step. ¹H NMR (300 MHz, CDCl₃) δ6.95(dd, J=15.7, 6.6 Hz, 1H), 5.77 (dd, J=15.7, 1.5 Hz), 3.72 (s, 3H), 2.44(m, 1H), 1.06 (d, J=6.7 Hz, 6H).

[0247] b. (2R,3S)-Methyl 2,3-dihydroxy-3-[isopropyl]propionate (XVIb;R¹=i-Pr; R³=Me): To a clear yellow solution of K₂OsO₂(OH)₄ (246.1 mg,0.67 mmol, 0.95 mol %), hydroquinine 1,4-phthalazinediyl diether (555.1mg, 0.71 mmol, 1.01 mol %), N-methylmorpholine N-oxide (50 wt % inwater, 25.0 mL, 0.106 mol, 1.51 equiv.), t-BuOH (84 mL), and H₂O (58 mL)was added at 25° C. the neat olefin XV (R¹=i-Pr; R³=Me) (9.0 g, 70.2mmol) via a syringe pump over a period of 48 h (the syringe wasconnected to tubing, whose tip was immersed in the solution throughoutthe reaction time). The resulting clear orange solution was then stirredfor another 60 min, after which time ethyl acetate (200 mL) and asolution of Na₂SO₃ (15.0 g) in H₂O (150 mL) were added, and theresulting mixture was stirred for 4 h. The phases were separated, andthe aqueous layer was extracted with more ethyl acetate (2×). Theorganic layers were then combined and the chiral ligand was extractedfrom the organic phase with a solution of 0.3 M H₂SO₄ in saturatedNa₂SO₄ (2×100 mL). The phases were once again separated and the aqueouslayer was extracted with more ethyl acetate (1×). The organic layerswere combined and dried over Na₂SO₄, filtered and concentrated in vacuo.This afforded 11.4 g (ca. 100%) of a white oily solid which was shown tobe 70% e.e (determined by ¹H NMR from a 1:1 molar solution of diol andEuropium tris[3-(heptafluoropropylhydroxymethylene)-(−)-camphorate] inC₆D₆). Recrystallisation from 35-60° C. petroleum ether afforded 6.8 g(60%) of (2R,3S)-Methyl 2,3-dihydroxy-3-[isopropyl]propionate (XVIb;R¹=i-Pr; R³=Me) that was ca. 100% e.e., obtained as white crystals,mp=32-34° C.; =−110.6° (c 1.04, CHCl₃)]. ¹H NMR (300 MHz, CDCl₃) δ4.28(dd, J=5.6, 1.8 Hz, 1H), 3.80 (s, 3H), 3.48 (m, 1H), 3.28 (m, 1H), 2.33(d, J=9.3 Hz, 1H), 1.87 (m, 1H), 1.02 (d, J=6.7 Hz, 3H), 0.95 (d, J=6.7Hz, 3H).

[0248] c. (2S,3S)-Methyl 2-bromo-3-dihydroxy-3-(isopropyl)propionate(XVIIb; R¹=i-Pr; R³=Me): (2R,3S)-Methyl 2,3-dihydroxy-3-[isopropyl]propionate XVIb (R¹=i-Pr; R³=Me) (30.0 g, 185.2 mmol) andtrimethylorthobenzoate (41.3 mL, 240.7 mmol) were dissolved in CH₂Cl₂(400 mL) and treated with BF₃.OEt₂ (1.16 mL, 9.25 mmol). After 2 h,triethylamine (1.8 mL, 13 mmol) was added, and the mixture wasconcentrated in vacuo and placed under full vacuum (0.05 mm Hg) for 70min. The residue was redissolved in CH₂Cl₂ (400 mL), cooled to 0° C. andtreated dropwise with acetyl bromide (14.3 mL, 194.5 mmol). After 2 h,additional acetyl bromide (0.68 mL, 9.25 mmol) was added. After 30 min,saturated NaHCO₃ solution (500 mL) was added and the mixture was stirredvigorously for 5-10 min. The layers were separated and the aqueous layerwas extracted with CH₂Cl₂ (2×20 mL). The combined organic layers weredried (Na₂SO₄), filtered and concentrated in vacuo affording the crudeα-bromo β-benzoate XVIIb (R¹=i-Pr; R³=Me) (66.23 g) as a clear colorlessoil, containing ˜9.3% by wt. methyl benzoate. For product: ¹H NMR (300MHz, CDCl₃) δ8.05-8.00 (m, 2H), 7.47-7.40 (m, 3H), 5.57 (dd, J=8.8, 3.9Hz, 1H), 4.47 (d, J=8.8 Hz, 1H), 3.67 (s, 3H), 2.45 (m, 1H), 1.01 (d,J=6.8 Hz, 6H).

[0249] d. (2R,3S)-Methyl2-azo-3-dihydroxy-3-[isopropyl]propionate(VIIIb; R¹=i-Pr; R³=Me): Sodium azide (24 g, 370 mmol) was added to 230mL of DMSO and the mixture was stirred at room temperature overnight. Tothe resultant solution was added a solution of (2S,3S)-Methyl2-bromo-3-dihydroxy-3-[isopropyl] propionate (XVIIb; R¹=i-Pr; R³=Me) (61g, 185 mmol) in 20 mL DMSO. After stirring for 11 h at room temperature,the mixture was poured into water (1.5 L) and ether (200 mL) and stirredvigorously for 10-15 min. Ether (100 mL) was added and the layers wereseparated. The aqueous layer was extracted with ether (2×100 mL) and thecombined organic layers were washed with water (2×100 mL) and brine (100mL), dried over MgSO₄, and concentrated in vacuo affording the crudeproduct (57.5 g), containing approximately 3% starting material and 8%elimination byproduct. For product: ¹H NMR (300 MHz, CDCl₃) δ8.07-8.02(m, 2H), 7.55-7.43 (m, 3H), 5.40 (dd, J=8.8, 2.8 Hz, 1H), 3.73 (s, 3H),2.24 (m, 1H), 1.04 (d, J=5.8 Hz, 3H), 0.98 (d, J=5.8 Hz, 3H).

[0250] e. Benzamide XIXb (R¹=i-Pr; R³=Me): To a cold (0-5° C.) solutionof (2R,3S)-Methyl 2-azo-3-dihydroxy-3-[isopropyl]propionate XVIIIb(R¹=i-Pr; R³=Me) (55 g) in methanol (300 mL) was added 94 mL of 4 MHCl/dioxane and 2.75 g of Pd(OH)₂/C. The mixture was purged withhydrogen and stirred at room temperature. The mixture was purged withhydrogen every 30 min to remove the liberated nitrogen. After 4 h, thereaction mixture was purged with nitrogen and additional Pd(OH)₂/C (1.3g) was added. The reaction mixture was purged with hydrogen and againpurged every hour for 4 h. The mixture was filtered and concentrated invacuo. The residue was dissolved in water and extracted with EtOAc. Theaqueous layer was basified with Na₂CO₃ and again extracted with EtOAc.The combined organic extracts were washed with brine, dried over Na₂SO₄,and concentrated to give a mixture of N- and O-benzoylated products,which was used directly in the next step.

[0251] f. trans-Oxazoline Ib (R¹=i-Pr; R³=Me): The crude product IXbobtained in Example 10e above (37.3 g, 141 mmol) was dissolved intoluene (350 mL). p-Toluenesulfonic acid (2.68 g, 14.1 mmol) was addedand the mixture was heated to reflux. Water was removed using a DeanStark trap. After 3 h, 2.5 mL of water had been collected. The reactionmixture was cooled, diluted with EtOAc (100 mL), washed successivelywith saturated NaHCO₃ (2×100 mL) and brine (100 mL), dried over MgSO₄,and concentrated. The residue was purified over a pad of silica gel(˜400 g), eluting with 25-30% EtOAc-hexanes to provide thetrans-oxazoline 1b (R¹=i-Pr; R³=Me). ¹H NMR (300 MHz, CDCl₃) δ8.01-7.97(m, 2H), 7.52-7.38 (m, 3H), 4.68 (apparent t, J=7 Hz, 1H), 4.57 (d, J=7Hz, 1H), 3.81 (s, 3H), 2.00-1.93 (m, 1H), 1.04 (d, J=6.7 Hz, 3H), 1.00(d, J=6.8 Hz, 3H).

EXAMPLE 12 Inactivation of Proteasome Activity

[0252] Purification of 20S proteasome and proteasome activator PA28 wasperformed as previously described (Dick et al., J. Biol. Chem. 271:7273(1996)).

[0253] 2 mL of assay buffer (20 mM HEPES, 0.5 mM EDTA, pH 8.0) andSuc-Leu-Leu-Val-Tyr-AMC in dimethyl sulfoxide were added to a 3 mLfluorescent cuvette, and the cuvette was placed in the jacketed cellholder of a Hitachi F-2000 fluorescence spectrophotometer. Thetemperature was maintained at 37° C. by a circulating water bath. 0.34mg of PA28 were added and the reaction progress was monitored by theincrease in fluorescence at 440 nm (λ_(ex)=380 nm) that accompaniesproduction of free AMC. The progress curves exhibited a lag phaselasting 1-2 min resulting from the slow formation of the 20S-PA28complex. After reaching a steady state of substrate hydrolysis,lactacystin was added to a final concentration of 1 mM, and the reactionwas monitored for 1 h. The fluorescence (F) versus time (t) data werecollected on a microcomputer using LAB CALC (Galactic) software.k_(inact) values were estimated by a nonlinear least-squares fit of thedata to the first order equation:

F=A(1−e ^(−kt))+C

[0254] where C=F=_(t=0) and A=F_(t=∞)−F_(t=0).

EXAMPLE 13 Inhibition of Intracellular Protein Degradation in C2C12Cells

[0255] C2C12 cells (a mouse myoblast line) were labeled for 48 hrs with³⁵S-methionine. The cells were then washed and preincubated for 2 hrs inthe same media supplemented with 2 mM unlabelled methionine. The mediawas removed and replaced with a fresh aliquot of the preincubation mediacontaining 50% serum, and a concentration of the compound to be tested.The media was then removed and made up to 10% TCA and centrifuged. TheTCA soluble radioactivity was counted. Inhibition of proteolysis wascalculated as the percent decrease in TCA soluble radioactivity. Fromthis data, an IC₅₀ for each compound was calculated.

EXAMPLE 14 Lactone Hydrolysis

[0256] The half-lives (t_(½)) for hydrolysis of β-lactone analogs to thecorresponding dihydroxy acids were measured at 37° C. at a concentrationof 200 mM in 20 mM HEPES, 0.5 mM EDTA, pH 7.8. Absorbance was measuredfor at least five half-lives (approximately 1 hour) at 230 nm, thewavelength at which there is the greatest difference in extinctioncoefficients for the lactone and dihydroxy. Half-lives were calculatedusing Guggenheim analysis (Gutfreund Enzymes: Physical Principles; Wileyand Sons: New York, 1975, pp 118-119). The results of Examples 12-14 arereported in Table 1. TABLE 1 Kinetics of Inhibition of 20S Proteasomeand Inhibition of Intracellular Protein Degradation

Compound R² Kobs/[I] M⁻¹ s⁻¹)^(a) IC₅₀ (μM)^(b) t_(1/2) min^(c) 2 Me20,000 0.7-1.1 13 3a Et 39,000 0.32 15.3 3b n-Pr 46,500 0.29 15.3 3cn-Bu 38,000 0.33 17 3d i-Bu 17,000 0.51 16.8 3e CH₂Ph 6,400 — 6.8 3f OMe82,200 86 3.7

[0257] The results indicate that the compounds of the present inventionare potent inhibitors of the proteasome.

EXAMPLE 15 Reduction of Infarct Size and Neuronal Loss

[0258] Methods

[0259] Male Sprague Dawley rats (250-400 g) were anesthetized withhaloethane and subjected to middle cerebral artery (MCA) occlusion usinga nylon filament for 2 h. Subsequently, the filament was removed andreperfusion of the infarcted tissue occurred for 24 hours before the ratwas sacrificed.

[0260] Immediately after the filament was withdrawn, the animals wereevaluated using a neurological scoring system. Neurological scores wereexpressed on a scale from 0 to 10, with 0 representing no neurologicaldeficit and 10 representing severe neurological deficit. After 24 hoursand before sacrifice, animals were evaluated a second time using thesame neurological scoring system.

[0261] Staining of coronal sections (2.0 mm×7-8) withtriphenyltetrazolium chloride (TTC) taken throughout the brain wereevaluated under blinded conditions using image analysis to determineinfarct size.

[0262] Dosing Regimen

[0263] Rats were given i.v. bolus injections (1.0 mL/kg) of eithervehicle (50% propylene glycol/saline; n=8) or7-n-propyl-clasto-lactacystin β-lactone (3b) (0.3 mg/kg; n=7) at 2 hoursafter the start of the occlusion. Two additional groups of rats weregiven i.v. bolus injections (1.0 mL/kg) of 3b at 0 minutes, 2 hours, and6 hours after the start of the occlusion. One group (0.1 mg/kg×3; n=6)received 0.1 mg/kg at each of these times, while another group (0.3mg/kg×3; n=7) received 0.3 mg/kg at each of the three timepoints.

[0264] Results

[0265] In animals treated with a single dose of7-n-propyl-clasto-lactacystin β-lactone (3b), infarct volume wasdecreased by 50% (FIG. 1, 0.3×1). Infarct volume was not significantlydecreased in either the 0.1 mg/kg×3 dosage group or the 0.3 mg/kg×3dosage group (FIG. 1).

[0266] All animals had a neurological score of 10±0 immediately afterthe 2 hour ischemic episode. At 24 hours, the vehicle-treated rats had amean score of 8.7±0.6, whereas rats treated with a single 0.3 mg/kg doseof 7-n-propyl-clasto-lactacystin β-lactone (3b) had a mean score of 4±1(FIG. 2). These data represent a 60% neurological improvement for thedrug-treated animals. No significant improvement in neurological scorewas observed in either the 0.1 mg/kg×3 dosage group of the 0.3 mg/g×3dosage group (FIG. 2).

[0267] Conclusion

[0268] 7-n-propyl-clasto-lactacystin β-lactone, given oncepost-ischemia, provides significant protection in both the degree ofneurological deficit and infarcted brain damage. From these preliminarydata, it appears that a single-dose regimen is preferred over amultiple-dose regimen.

[0269] Having now fully described this invention, it will be understoodto those of ordinary skill in the art that the same can be performedwithin a wide and equivalent range of conditions, formulations, andother parameters without affecting the scope of the invention or anyembodiment thereof. All patents and publications cited herein are fullyincorporated by reference herein in their entirety.

What is claimed is:
 1. A process for forming a γ-lactam carboxylic acidof Formula V:

or a salt thereof, wherein R¹ is alkyl, alkenyl, alkynyl, cycloalkyl,aryl, alkaryl, aralkyl, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; R² is alkyl,cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, oramido, where the ring portion of any of said aryl, aralkyl, or alkarylcan be optionally substituted; and said process comprising: (a)deprotonating a substituted aryl or heteroaryl oxazoline of Formula I:

where R¹ is as defined above; R³ is alkyl, alkenyl, alkynyl, cycloalkyl,aryl, alkaryl, any of which can be optionally substituted; and R⁴ isaryl or heteroaryl, either of which may be optionally substituted; bytreating said substituted aryl or heteroaryl oxazoline with a strongbase to form an enolate; (b) transmetallating said enolate with a metalselected from the group consisting of titanium, aluminum, tin, zinc,magnesium and boron, and thereafter treating with a formyl amide ofFormula XIV:

where R² is as defined above, and R⁵ and R⁶ are independently one ofalkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogenatom to which they are attached form a 5- to 7-membered heterocyclicring, which may be optionally substituted, and which optionally mayinclude an additional oxygen or nitrogen atom, to form an adduct ofFormula II:

where R¹ through R⁶ are as defined above; c) catalytically hydrogenatingsaid adduct of Formula II to form a γ-lactam of Formula IV.

where R¹, R² and R³ are as defined above; and d) saponifying saidγ-lactam of Formula IV to form a carboxylic acid of Formula V.
 2. Theprocess of claim 1, further comprising treating the carboxylic acid ofFormula V with a cyclizing reagent to form a clasto-lactacystinβ-lactone of Formula VII:

wherein R¹ and R² are as defined in claim
 1. 3. The process of claim 2,wherein said cyclizing is effected with a reagent selected from thegroup consisting of aryl sulfonyl chlorides,benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate,O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborateand alkyl-aryl- or alkenyl chlorofomates.
 4. The process of claim 2,further comprising reacting the clasto-lactacystin β-lactone of FormulaVII with a thiol, R⁷SH, to form lactacystin having Formula VI:

wherein R¹ and R² are as defined in claim 1; and R⁷ is alkyl, aryl,aralkyl, alkaryl, wherein any of said alkyl, aryl, aralkyl or alkarylcan be optionally substituted.
 5. The process of claim 4, whereinclasto-lactacystin β-lactone is converted to lactacystin by treating theβ-lactone with N-acetylcysteine.
 6. The process of claim 1, wherein thecarboxylic acid intermediate of Formula V is directly coupled to athiol, R⁷SH, to form a lactacystin having Formula VI:

wherein R¹ and R² are as defined in claim 1; and R⁷ is alkyl, aryl,aralkyl, alkaryl, wherein any of said alkyl, aryl, aralkyl or alkarylcan be optionally substituted.
 7. The process of claim 1, wherein instep (a) said strong base is selected from the group consisting ofhindered amide bases; alkali metal hexamethyldisilazides; or hinderedalkyllithium reagents.
 8. The process of claim 1, wherein in step (a)the reaction is conducted at reduced temperature in an ethereal solvent.9. The process of claim 8, wherein in step (a) said ethereal solvent isselected from the group consisting of diethyl ether, tetrahydrofuran,and dimethoxyethane, and said reaction temperature is from about −100°C. to about −30° C.
 10. The process of claim 1, wherein in step (b) saidenolate is transmetallated with titanium or aluminum or a mixturethereof.
 11. The process of claim 1, wherein in step (b), said enolateis transmetallated by reaction with Me₂AlCl.
 12. The process of claim10, wherein between one and three molar equivalents of said metal areused.
 13. The process of claim 1, wherein in step (c) said catalytichydrogenolysis of the adduct II, affords the desired γ-lactam (IV) as amixture with an aminodiol III:

wherein R¹-R⁶ are as defined in claim
 1. 14. The process of claim 13,wherein said hydrogenolysis is conducted in the presence of a catalystselected from the group consisting of palladium black, palladium onactivated carbon, and palladium hydroxide on carbon; and in the presenceof an organic solvent selected from the group consisting of loweralkanols, lower alkanoates, lower alkanoic acids and mixtures thereof.15. The process of claim 14, wherein said organic solvent is selectedfrom the group consisting of methanol, ethanol, isopropanol, ethylacetate, acetic acid, and mixtures thereof.
 16. The process of claim 13,wherein the crude product mixture is heated to convert aminodiol III tothe γ-lactam IV.
 17. The process of claim 1, wherein R¹ is C₁₋₁₂alkyl,C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkylor C₁₋₆alk(C₆₋₁₀)aryl; R² is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl,C₂₋₈ alkynyl C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl; R³ isC₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl, C₆₋₁₀ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl; R⁴ is C₆₋₁₀aryl, or a heteroarylgroup selected from the group consisting of thienyl, benzo[b]thienyl,furyl, pyranyl, isobenzofuranyl, benzoxazolyl, 2H-pyrrolyl, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,4H-quinolizinyl, isoquinolyl, quinolyl, or triazolyl; and R⁵ and R⁶ areindependently C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl ortogether with the nitrogen atom to which they are attached form a 5- to7-membered heterocycle which can be optionally substituted, and whichoptionally can include an additional oxygen or nitrogen atom.
 18. Theprocess of claim 17, wherein R¹ is C₁₋₆alkyl, C₃₋₆cycloalkyl, orC₆₋₁₀aryl; R² is methyl, ethyl, propyl, butyl, methoxy, or ethoxy; R³ ismethyl, ethyl, tert-butyl or benzyl; R⁴ is phenyl or phenyl substitutedby halogen, C₁₋₆alkyl, C₁₋₆alkoxy, carboxy, or amino; and NR⁵R⁶ is oneof dimethylamino, diethylamino, pyrrolidino, piperidino, morpholino,oroxazolidinone substituted by halogen, C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkyl,C₁₋₆alkoxy, carboxy, or amino.
 19. A process for forming a substitutedoxazoline compound of Formula II:

said method comprising: (a) deprotonating a substituted aryl orheteroaryl oxazoline of Formula I:

by treating said substituted aryl or heteroaryl oxazoline with a strongbase to form an enolate; and (b) transmetallating said enolate with ametal selected from the group consisting of titanium, aluminum tin,zinc, magnesium and boron, and thereafter reacting with a formyl amideof Formula XIV:

wherein for each of Formulae I, II and XIV: R¹ is alkyl, alkenyl,alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion ofany of said aryl, aralkyl, or alkaryl can be optionally substituted; R²is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy,alkoxyalkyl, or amido, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; R³ is alkyl, alkenyl,alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionallysubstituted; R⁴ is optionally substituted aryl or optionally substitutedheteroaryl; and R⁵ and R⁶ are independently one of alkyl or alkaryl; orR⁵ and R⁶ when taken together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocyclic ring, which can beoptionally substituted, and which optionally include an additionaloxygen or nitrogen atom.
 20. The process of claim 19, further comprisingcatalytically hydrogenating said oxazoline compound of Formula II, andthereafter optionally refluxing the resulting reaction mixture, wherebya β-lactam of Formula IV is formed:

wherein R¹, R² and R³ are as defined in claim
 19. 21. The process ofclaim 20, further comprising saponifying a compound of Formula IV andthereafter cyclizing to form a clasto-lactacystin β-lactone compoundhaving Formula VII:

wherein R¹ and R² are as defined in claim
 19. 22. A process for forminga substituted aryl oxazoline compound of Formula Ia:

wherein R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl,aralkyl, where the ring portion of said aryl, aralkyl, or alkaryl can beoptionally substituted; R³ is alkyl, cycloalkyl, aryl, alkaryl, any ofwhich can be optionally substituted; and R⁴ is optionally substitutedaryl or optionally substituted heteroaryl; said method comprising: (a)asymmetrically dihydroxylating an alkene intermediate of Formula XV:

to form an optically active diol of Formula XVIa:

(b) reacting said optically active diol of Formula XVIa with anorthoester under acid catalysis to give a mixed orthoester, andthereafter reacting the resulting mixed orthoester intermediate with areagent selected from the group consisting of acyl halides, HCl, HBr,HI,Me₃SiCl, Me₃SiI, Me₃SiBr and halogen-containing Lewis acids to form ahaloester derivative of Formula XVIIa:

wherein X is Cl, Br, or I; (c) reacting said haloester derivative withan alkali metal azide to form an azide of Formula XVIIIa:

(d) hydrogenating said azide to form a compound of Formula XIXa:

(e) subjecting the compound of Formula XIXa to ring closing conditionsto form said substituted phenyloxazoline of Formula Ia; wherein for eachof Formulae XVa, XVIA, XVIIa, XVIIIa and XIXa, R¹, R³ and R⁴ are asdefined above for Formula Ia.
 23. The process of claim 22, wherein instep (a) the dihydroxylation reaction is conducted with AD-mix-β in thepresence of methane sulfonamide to stereoselectively afford the diol ofFormula XVIa.
 24. The process of claim 22, wherein in step (a) thedihydroxylation reaction is conducted using an N-oxide as a reoxidant.25. The process of claim 22, wherein in step (b) said diol of FormulaXVIa is treated with an orthoester under Lewis or Brönsted acidcatalysis to give a mixed orthoester, which is converted in situ to ahaloester of Formula XVIIa wherein X is Br by treatment with acetylbromide.
 26. The process of claim 25, wherein the orthoester employed inthis reaction is an aromatic carboxylic acid orthoester.
 27. The processof claim 26, wherein the orthoester is trimethyl orthobenzoate.
 28. Theprocess of claim 25, wherein said acid catalyst is HBr, SnCl₄, TiCl₄,BBr₃ or boron trifluoride.
 29. The process of claim 22, wherein in step(c) crude haloester of Formula XVIIa is converted to the azide ofFormula XVIIIa by treatment with an alkali metal azide in a polaraprotic organic solvent.
 30. The process of claim 22, wherein in step(d) said catalytic hydrogenation of the azide of Formula XVIIIa isconducted over a palladium catalyst in ethyl acetate.
 31. The process ofclaim 30, wherein said catalytic hydrogenation proceeds with concomitantmigration of the aroyl group to afford the hydroxyamide of Formula XIXa.32. The process of claim 22, wherein in step (e) the hydroxyamide ofFormula XIXa is treated with thionyl chloride in methylene chloride toeffect ring closure with inversion of the hydroxyl to produce thecis-substituted oxazoline of Formula Ia.
 33. The process of claim 32,wherein the cis-oxazoline is converted to the trans-oxazoline underequilibrating condition by inversion of configuration of the estersubstituents.
 34. A process for forming a substituted aryl oxazolinecompound of Formula Ib:

wherein R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl,aralkyl, where the ring portion of said aryl, aralkyl, or alkaryl can beoptionally substituted; R³ is alkyl, cycloalkyl, aryl, alkaryl, any ofwhich can be optionally substituted; and R⁴ is optionally substitutedaryl or optionally substituted heteroaryl; said method comprising: (a)asymmetrically dihydroxylating an alkene intermediate of Formula XV:

to form an optically active diol of Formula XVIb:

(b) reacting said optically active diol of Formula XVIb with anorthoester under acid catalysis to give a mixed orthoester, andthereafter reacting the resulting mixed orthoester intermediate with areagent selected from the group consisting of acyl halides, HCl, HBr,HI,Me₃SiCl, Me₃SiI, Me₃SiBr and halogen-containing Lewis acids to form ahaloester derivative of Formula XVIIb:

wherein X is Cl, Br, or I; (c) reacting said haloester derivative withan alkali metal azide to form an azide of Formula XVIIIb:

(d) hydrogenating said azide to form a compound of Formula XIXb:

(e) subjecting the compound of Formula XIXb to ring closing conditionsto form said substituted phenyloxazoline of Formula Ib; wherein for eachof Formulae XVb, XVIb, XVIIb, XVIIIb and XIXb, R¹, R³ and R⁴ are asdefined above for Formula Ib.
 35. The process of claim 34, wherein instep (a) the dihydroxylation reaction is conducted with AD-mix-α in thepresence of methane sulfonamide to stereoselectively afford the diol ofFormula XVIb.
 36. The process of claim 34, wherein in step (a) thedihydroxylation reaction is conducted using an N-oxide as a reoxidant.37. The process of claim 34, wherein in step (b) said diol of FormulaXVIb is treated with an orthoester under Lewis or Brönsted acidcatalysis to give a mixed orthoester, which is converted in situ to ahaloester of Formula XVIIb wherein X is Br by treatment with acetylbromide.
 38. The process of claim 37, wherein the orthoester employed inthis reaction is an aromatic carboxylic acid orthoester.
 39. The processof claim 38, wherein the orthoester is trimethyl orthobenzoate.
 40. Theprocess of claim 37, wherein said acid catalyst is HBr, SnCl₄, TiCl₄,BBr₃ or boron trifluoride.
 41. The process of claim 34, wherein in step(c) crude haloester of Formula XVIIb is converted to the azide ofFormula XVIIIb by treatment with an alkali metal azide in a polaraprotic organic solvent.
 42. The process of claim 34, wherein in step(d) said catalytic hydrogenation of the azide of Formula XVIIIb isconducted over a palladium catalyst in ethyl acetate.
 43. The process ofclaim 42, wherein said catalytic hydrogenation proceeds with concomitantmigration of the aroyl group to afford the hydroxyamide of Formula XIXb.44. The process of claim 34, wherein in step (e) the hydroxyamide ofFormula XIXb is treated with thionyl chloride in methylene chloride toeffect ring closure with inversion of the hydroxyl to produce thetrans-substituted oxazoline of Formula Ib.
 45. A process for forming acompound of Formula XIXa:

said method comprising: hydrogenating an azide compound having FormulaXVIIIa:

wherein for each of Formulae XIXa and XVIIIa: R¹ is alkyl, alkenyl,alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion ofany of said aryl, aralkyl, or alkaryl can be optionally substituted; R³is alkyl, cycloalkyl, aryl, alkaryl, any of which can be optionallysubstituted; and R⁴ is aryl or heteroaryl, either of which may beoptionally substituted.
 46. The process of claim 45, further comprisingsubjecting a compound of Formula XIXa to ring closing conditions to forma substituted oxazoline compound of Formula Ia:

wherein R¹, R³ and R⁴ are as defined in claim
 45. 47. A process forforming a compound of Formula XIXb:

said method comprising: hydrogenating an azide compound having FormulaXVIIIb:

wherein for each of Formulae XIXb and XVIIIb: R¹ is alkyl, alkenyl,alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion ofany of said aryl, aralkyl, or alkaryl can be optionally substituted; R³is alkyl, cycloalkyl, aryl, alkaryl, any of which can be optionallysubstituted; and R⁴ is aryl or heteroaryl, either of which may beoptionally substituted.
 48. The process of claim 47, further comprisingsubjecting a compound of Formula XIXb to ring closing conditions to forma substituted oxazoline compound of Formula Ib:

wherein R¹, R³ and R⁴ are as defined in claim
 47. 49. A compound ofFormula VI or VII:

or a salt thereof, wherein: R¹ is C₁₋₁₂alkyl, C₃₋₈cycloalkyl,C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl; R² is C₂₋₆alkyl; and R⁷ is alkyl, aryl, aralkyl,alkaryl, wherein any of said alkyl, aryl, aralkyl or alkaryl can beoptionally substituted.
 50. A compound of claim 49, wherein R¹ is C₁₋₄alkyl.
 51. A compound of claim 50, wherein R¹ is isopropyl.
 52. Acompound of claim 49, wherein R² is ethyl, n-propyl, n-butyl orisobutyl.
 53. A compound of claim 52, wherein R² is ethyl.
 54. Acompound of claim 52, wherein R² is n-propyl.
 55. A compound of claim52, wherein R² is n-butyl.
 56. A compound of claim 52, wherein R² isisobutyl.
 57. An enantiomerically-enriched formyl amide of Formula XIV:

or a salt thereof, wherein R² is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl,C₂₋₈alkynyl, C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl; andR⁵ and R⁶ are independently C₁₋₆ alkyl, C₆₋₁₀ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl, or together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocycle which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom.
 58. A pharmaceutical composition comprising a compoundaccording to any one of claims 49-56 and a pharmaceutically acceptablecarrier or diluent.
 59. A compound of Formula II:

or a salt thereof, wherein R¹ is alkyl, alkenyl, alkynyl, cycloalkyl,aryl, alkaryl, aralkyl, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; R² is alkyl,cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, oramido, where the ring portion of any of said aryl, aralkyl, or alkarylcan be optionally substituted; R³ is alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkaryl, any of which can be optionally substituted;R⁴ is optionally substituted aryl or optionally substituted heteroaryl;and R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶when taken together with the nitrogen atom to which they are attachedform a 5- to 7-membered heterocyclic ring, which can be optionallysubstituted, and which optionally include an additional oxygen ornitrogen atom.
 60. A compound of claim 59, wherein R¹ is C₁₋₁₂alkyl,C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkylor C₁₋₆alk(C₆₋₁₀)aryl, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; R² is C₁₋₈alkyl,C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkylor C₁₋₆alk(C₆₋₁₀)aryl, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; R³ is C₁₋₈alkyl,C₃₋₈cycloalkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl, C₆₋₁₀ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, any of which can be optionallysubstituted; R⁴ is optionally substituted C₆₋₁₀aryl, or an optionallysubstituted heteroaryl group selected from the group consisting ofthienyl, benzo[β]thienyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, ortriazolyl; and R⁵ and R⁶ are independently C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkylor C₁₋₆alk(C₆₋₁₀)aryl or together with the nitrogen atom to which theyare attached form a 5- to 7-membered heterocycle which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom.
 61. A compound of Formula III:

or a salt thereof wherein: R¹ is alkyl, alkenyl, alkynyl, cycloalkyl,aryl, alkaryl, aralkyl, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; R² is alkyl,cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, oramido, where the ring portion of any of said aryl, aralkyl, or alkarylcan be optionally substituted; R³ is alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkaryl, any of which can be optionally substituted;and R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶when taken together with the nitrogen atom to which they are attachedform a 5- to 7-membered heterocyclic ring, which can be optionallysubstituted, and which can optionally include an additional oxygen ornitrogen atom.
 62. A compound of claim 61, wherein R¹ is C₁₋₁₂alkyl,C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl, C₆₋₁₀ ar(C₁₋₆)alkylor C₁₋₆alk(C₆₋₁₀)aryl, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; R² is C₁₋₈alkyl,C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl, C₆₋₁₀ar (C₁₋₆)alkylor C₁₋₆alk(C₆₋₁₀)aryl, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; and R³ is C₁₋₈alkyl,C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl, C₆₋₁₀ar (C₁₋₆)alkylor C₁₋₆alk(C₆₋₁₀)aryl, any of which can be optionally substituted; andR⁵ and R⁶ are independently C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl, or together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocycle which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom.
 63. A compound of Formula XVIIa or XVIIb:

or a salt thereof, wherein R¹ is alkyl, alkenyl, alkynyl, cycloalkyl,aryl, alkaryl, aralkyl, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; R² is Cl, Br or I; R³is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which canbe optionally substituted; and R⁴ is optionally substituted aryl oroptionally substituted heteroaryl.
 64. A compound of Formula XVIIIa orXVIIIb

or a salt thereof, wherein R¹ is alkyl, alkenyl, alkynyl, cycloalkyl,aryl, alkaryl, aralkyl, wherein the ring portion of any of said aryl,aralkyl or alkaryl can be optionally substituted; R³ is alkyl, alkenyl,alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionallysubstituted; and R⁴ is optionally substituted aryl or optionallysubstituted heteroaryl.
 65. A process for forming anenantiomerically-enriched formyl amide of Formula XIV:

or a salt thereof, wherein R² is alkyl, cycloalkyl, aryl, alkaryl,aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portionof any of said aryl, aralkyl, or alkaryl can be optionally substituted;R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ whentaken together with the nitrogen atom to which they are attached form a5- to 7-membered heterocyclic ring, which can be optionally substituted,and which optionally include an additional oxygen or nitrogen atom; saidmethod comprising: (a) acylating an anion of a compound of Formula VIII:

where R⁸ is isopropyl or benzyl, with R²CH₂COCl to form anacyloxazolidinone of Formula IX:

where R² and R⁸ are as defined above; (b) stereoselectively reacting theacyloxazolidinone of Formula IX with benzyloxymethyl chloride to form aprotected alcohol of Formula X:

where R² and R⁸ are as defined above; (c) hydrolyzing the protectedalcohol of Formula X to form a carboxylic acid of Formula XI:

where R² is as defined above; (d) coupling said acid of Formula XI withan amine R⁵R⁶NH₂ to provide an amide of Formula XII:

where R², R⁵ and R⁶ are as defined above; (e) catalyticallyhydrogenating, the amide of Formula XII to form an alcohol of FormulaXIII:

where R², R⁵ and R⁶ are as defined above; and (f) oxidizing theresultant alcohol of Formula XIII to give a formyl amide of Formula XIV.66. The process of claim 65, wherein: R² is C₁₋₈alkyl, C₃₋₈cycloalkyl,C₂₋₈alkenyl, C₂₋₈alkynyl C₆₋₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl; and R⁵ and R⁶ are independently C₁₋₆alkyl,C₆₋₁₀ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl or together with the nitrogenatom to which they are attached form a 5- to 7-membered heterocyclewhich can be optionally substituted, and which optionally can include anadditional oxygen or nitrogen atom.
 67. A method of inhibitingproteasome function in a cell, comprising contacting said cell with acompound of claim
 49. 68. A method of inhibiting proteasome function ina mammal, comprising administering to said mammal a compound of claim 49in an amount effective to inhibit proteasome function.
 69. A method oftreating inflammation, comprising administering to a subject aneffective anti-inflammatory amount of a compound of claim
 49. 70. Amethod of treating cancer, comprising administering to a subject aneffective antitumor or antimetastic amount of a compound of claim 49.71. A method of treating ischemic or reperfusion injury in a mammalcomprising administering to said mammal an effective amount of acompound of claim
 49. 72. The method of claim 71, wherein the ischemiais the result of vascular occlusion.
 73. The method of claim 71, whereinsaid vascular occlusion occurs during a stroke.